Optical Control of the Cardiac Rhythm with Photoswitchable NaV1.5 Channel Blockers

Optical Control of the Cardiac Rhythm with Photoswitchable NaV1.5 Channel Blockers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Optical Control of the Cardiac Rhythm with Photoswitchable NaV1.5 Channel Blockers Zhuo Huang, Shiqi Liu, Weiqiang Guan, Zhangqiang Li, Wei Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6532136/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Voltage-gated sodium channel Na V 1.5 is essential for cardiac excitability, mediating the rapid depolarization phase of the cardiac action potential (AP) and ensuring proper electrical conduction in the heart. Dysfunction of Na V 1.5 is implicated in life-threatening arrhythmias, making it a critical therapeutic target. Acting as a Na V 1.5 open-state blocker, quinidine demonstrates efficacy in arrhythmia treatment, but its low specificity restricts its clinical application. Here, we reported an optopharmacological strategy which enables a precise and optical control of Na V 1.5 function by means of photoswitchable quinidine derivatives. Through systematic structural optimization, we identified azo-Q2a as a high-performance photoswitchable inhibitor, exhibiting low activity in the dark or under 480 nm light irradiation ( trans isomer), while approximately 7-fold higher efficacy was observed under 365 nm light irradiation ( cis isomer). Of note, azo-Q2a demonstrated exceptional selectivity for Na V 1.5 over other cardiac ion channels, minimizing potential off-target effects. Furthermore, by solving the cryo-EM structure of the Na V 1.5 in complex with the cis -active isomer azo-Q2a (3.1 Å resolution), we revealed the essential binding site that is responsible for the optical control of Na V 1.5. Finally, azo-Q2a also attenuates heart rate of living zebrafish larvae with light, showing its potential in cardiac related research and treatment. Our work not only establishes azo-Q2a as a robust photoswitchable inhibitor for Na V 1.5 but also provides a structural blueprint for the rational design of next-generation optopharmacological antiarrhythmic agents. Biological sciences/Chemical biology/Ion channels Health sciences/Diseases/Cardiovascular diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The Na V 1.5 encoded by the SCN5A gene is the predominant subtype of voltage-gated sodium channels family (Na V 1.1-1.9) in the heart, which plays an essential role in the excitability of cardiomyocytes 1 . The channels mediate the sodium influx into cells resulting in inward sodium current ( I Na ), which induces fast depolarization of rapid response cardiac action potential (AP). Therefore, dysfunction of Na V 1.5 channels underlies multiple cardiac channelopathies 2–4 , including long QT syndrome type 3 (LQT3), Brugada syndrome (BrS), sick sinus syndrome (SSS), dilated cardiomyopathy (DCM), atrial fibrillation (AF), and progressive cardiac conduction disease (PCCD). This underscores its status as a pivotal therapeutic target for rhythm management. Additionally, given its pivotal role in action potential generation and conduction, Na V 1.5 has long been recognized as a canonical pharmacological target for Class I antiarrhythmic drugs. Among them, quinidine belongs to the Ⅰ a subclass as the first antiarrhythmic drug in the world. Acting as a Na V 1.5 channel open state blocker, quinidine inhibits peak I Na , thereby reducing the membrane excitability of cardiomyocytes, slowing down the depolarization and conduction velocity of rapid response cardiac AP. It has been previously used for conditions such as atrial premature beats (APB), paroxysmal supraventricular tachycardia (PSVT), AF and LQT3 5 . However, its therapeutic utility is severely constrained by extensive off-target polypharmacology. Quinidine often accompanies inhibition of hERG channel, thereby prolonging APD, ERP and QT interval 6,7 , which increases its off-target activities 8 . Furthermore, quinidine exhibits promiscuous binding to multiple extracardiac receptors, including nicotinic acetylcholine receptors (nAChRs) 9 , 5-hydroxytryptamine receptors 3 (5-HT 3 ) 10 and α adrenergic receptors 11 . Its nonspecific pharmacodynamic profile induces intolerable gastrointestinal (nausea, diarrhea) and neurological (tinnitus, cinchonism) adverse effects 12 in approximately 20–30 percent 5 of patients during clinical application. These limitations underscore the imperative for developing targeted Na V 1.5 modulators with improved specificity. Light has emerged as a powerful biophysical modality for spatiotemporally precise control of biological systems 13–15 . Through its integration with pharmacology, optopharmacology enables non-invasive and rapidly reversible modulation of target-specific receptors and ion channels. This innovative strategy effectively circumvents the problems of off-target and dose-limiting adverse effects of traditional drugs, and thus achieve a much more precise treatment. Moreover, optopharmacology has been widely applied in ion channels 16–18 , transporters 19 , G protein-coupled receptors 20,21 , receptor-linked enzymes 22 . Herein, we describe a photoactivated quinidine derivative with a photoswitchable azobenzene moiety incorporating, which enables reversible optical control over Na V 1.5 function and heart rate. We further characterize the binding sites of our lead compound cis - azo-Q2a using single-particle electron cryo-microscopy (cryo-EM). This provides a structural template for achieving selectivity and facilitate rational photoswitchable drug design. Results Rational design and synthesis of photoswitchable quinidine derivatives As disclosed in the recently cryo-structure of Na V 1.5 ion channel in complex with quinidine 23 , quinidine blocks the Na V 1.5 channel mainly with the quinoline moiety. While the quinuclidine group directly interrupts the central permeation pathway, the quinolone aromatic ring primarily interacts with the F1760 residue through a π-π stacking interaction. Much inspired by the above-mentioned crucial interaction, we hypothesized that quinidine derivatives with additional aryl substituents attached to the quinolone core would be beneficial for the binding affinity of Na V 1.5 ion channel. Initially, we designed three simple quinidine phenyl derivatives with phenyl substituents at the C-2 ( Q1 ), C-3 ( Q2 ), and C-6 ( Q3 ) positions of the quinoline core (Fig. 1 b). Specifically, Q1 was synthesized from quinidine via a Minisci reaction 24 while Q2 and Q3 were prepared through Suzuki-Miyaura coupling reactions with phenylboronic acid using 3-I-QD and 6-OTf-QD as starting materials, respectively 25,26 . Remarkably, whole-cell patch-clamp recordings in HEK293T cells heterologously expressing Na V 1.5 channels revealed that Q1 and Q3 exhibited significantly higher inhibition rates than quinidine at 10 µM concentration (Fig. 1 a). These results strongly suggest that enhanced π-π stacking interactions may strengthen the binding between quinidine derivatives and the Na V 1.5 ion channel. Next, we explored photoreactive quinidine derivatives, focusing on azobenzene-based chromophores due to their widespread application. In total, nine azo-quinidine derivatives ( azo-QD s) were designed, featuring azobenzene moieties installed at ortho-, meta-, and para-positions with phenyl spacers at the C-2, C-3, and C-6 positions of the quinoline core (Fig. 1 a). Notably, our synthetic approach for azo-QD s utilized Suzuki-Miyaura coupling with three key intermediates: 3-I-QD , 6-OTf-QD , and 2-Br-QD (Scheme 1 ). The synthesis of 2-Br-QD began with the oxidation of quinidine using m -CPBA, followed by reduction with NaHSO 3 , yielding N -oxide QD in 45% over two steps 27 . Subsequent bromination via the Yu protocol afforded 2-Br-QD in 46% yield 28 . For 3-I-QD , direct derivatization was achieved through Suzuki-Miyaura cross-coupling with p -azo-BPin and m -azo-BPin, producing the desired products in 27% and 24% yields, respectively. Meanwhile, 6-OTf-QD and 2-Br-QD were successfully coupled to furnish target compounds in 86%, 48%, 38%, and 33% yields. However, attempts to employ sterically hindered o -azo-BPin in the Suzuki-Miyaura reaction failed to yield any quinidine derivatives. Photochemical characterization of quinidine azobenzene derivatives With all 6 quinidine azobenzene derivatives in hand, photochemical characterization has been explored (Fig. 2 a and Supplementary Fig. 1). Using UV-Vis spectroscopy, we characterized the spectral properties of all cis - and trans - quinidine azobenzene derivatives (Fig. 2 b and Supplementary Fig. 1). In the dark, they existed as the thermodynamically favored trans isomer. Isomerization to the cis isomer was triggered upon UV-A irradiation (365 nm), and this process was reversed within 5 seconds upon exposure to blue light (480 nm). Taken azo-Q2a as an example, we observed a rapid and reversable photoswitch upon oscillating between 365 and 480 nm illumination over cycles (Fig. 2 c). Using 1 H-NMR spectroscopy, we assessed the relative concentrations of the two isomers in equilibrium after 365 and 480 nm illumination (Fig. 2 d). After illumination at 365nm, trans isomer azo-Q2a was efficiently isomerized to cis isomer (86%); 480 nm illumination resulted in efficient isomerization back to the trans isomer (79%). Finally, we monitored thermal relaxation of the cis - azo-Q2a in aqueous solution at room temperature: the half-life for relaxation from the cis to the trans isomer was 107h (Fig. 2 e). Optical control of Na1.5 channels with photoswitchable quinidine azobenzene derivatives To determine whether quinidine azobenzene derivatives support optical control of Na V 1.5 channels, we initially profiled the dose-dependent inhibition activities of quinidine and six derivatives to Na V 1.5 current in the dark or under 365nm illumination, using whole-cell patch-clamping recordings in HEK293T cells heterologously expressing Na V 1.5 channels. (Fig. 3 a-d and Supplementary Fig. 2). When holding the cellular membrane potential at -120 mV, the expected Na + currents were observed at the typical voltage steps 29 (-120 to -20 mV). All of the examined quinidine azobenzene derivatives in the cis isomer conferred stronger inhibition activity than quinidine, and azo - 2P , azo-Q2a , and azo - 6M displayed a significant increase in their IC 50 values after 365 nm illumination. (Fig. 3 a-c and Supplementary Fig. 2). Among the examined quinidine azobenzene derivatives, azo-Q2a showed the most prominent alteration (IC 50 = 11.76 µM in cis ; 81.85 µM in trans , Fig. 3 c), while quinidine had no obvious difference (IC 50 = 35.87 µM under 365 nm; 41.21 µM in dark, Fig. 3 d). IC 50 values are detailed in Supplementary Table 1. We measured current-voltage ( I - V ) steps between − 120 and + 40 mV (5 mV steps) in the presence of 10 µM azo-Q2a (Fig. 3 e). In its cis isomer, azo-Q2a strongly suppressed inward currents conducted by Na V 1.5 channels, whereas the trans isomer had no discernable effect. In addition, neither isomer altered the activation property of Na V 1.5, assessed as conductance-voltage ( g - V ) relationships and the V 50 value (Fig. 3 f, Supplementary Table 2). Note that blockade of Na V 1.5 channels was readily reversible as we repeatedly photoswitched azo-Q2a between its trans and cis isomer (Fig. 3 g-h). azo-Q2a is highly selective and significantly reduces hERG toxicity of quinidine Recalling that quinidine’s off-target interactions that inhibit hERG channels lead to serious cardiotoxicity 7 , we investigated the potential inhibition activity of azo-Q2a towards other ion channels known to contribute to cardiac action potential, including K V 4.3 ( I to ), K ir 2.1 ( I K1 ), hERG ( I Kr ) and Ca V 1.2 ( I CaL ) channels (Fig. 4 a). The experiments monitored the currents of these channels under both 365 and 480 nm illumination. Currents of these ion channels were recorded using whole-cell patch-clamping in HEK293T and CHO cell lines expressing cardiac ion channels (see Methods section for details). The inhibition rate of 10 µM azo-Q2a for Na V 1.5 channels was significantly different between the cis (61.1 ± 4.8%) and trans (2.5 ± 1.6%) isomers, whereas both azo-Q2a isomers exerted in same inhibition activity against K V 4.3, K ir 2.1, hERG and Ca V 1.2 channels (Fig. 4 b). Quantification of the inhibition showed that azo-Q2a endows light sensitivity only to Na V 1.5 but has negligible effects on K V and Ca V channels, yielding photoswitching efficacies of 33.1 ± 4.3% for Na V 1.5 and far less for the rest channels, of 5.2 ± 1.7% (K V 4.3), 3.6 ± 4.2% (K ir 2.1), 7.9 ± 4.5% (hERG), 7.7 ± 5.4% (Ca V 1.2) (Fig. 4 h and Supplementary Table 4). To compare the difference in hERG channel inhibition activity between azo-Q2a and quinidine, we repeated these assessments through application of 10 and 30 µM quinidine. As expected, quinidine showed comparable pharmacological behavior across all tested ion channels without optical control property (Fig. 4 b and Supplementary Fig. 3a-c). Notably, at the same concentration (10 µM), cis - azo-Q2a exhibited approximately 7-fold higher Na V 1.5 inhibition compared to quinidine, while its hERG inhibition activity was roughly half that of quinidine (Fig. 4 b and Supplementary Table 3). In addition, we further evaluated the dose-response relationship of azo-Q2a and quinidine on hERG channels under 365 and 480 nm illumination, which showed that the IC 50 value of azo-Q2a on hERG was 16-fold lower than that of quinidine (Supplementary Fig. 3d). Together, these results demonstrate that azo-Q2a possesses high selectivity for Na V 1.5 channels among cardiac ion channels, significantly reducing the hERG channel inhibition activity observed with quinidine. This characteristic makes azo-Q2a a promising candidate for the development of photoactivated antiarrhythmic therapies. azo-Q2a reversibly inhibits Na + currents in rat primary cardiomyocytes We next investigated azo-Q2a as an agent for the selective optical control of Na V 1.5 channels in rat primary cardiomyocytes. We recorded voltage-gated Na + and K + currents from primary ventricular myocytes under 365 and 480 nm illumination (Fig. 4 c). Consistent with our observations in the transient transfection cell experiments, Na + current in cardiomyocytes was reduced at 365 nm illumination after the addition of azo-Q2a (10 µM), and this was reversed by blue light (480 nm) (Fig. 4 d). The selective optical control of Na V 1.5 channels was over multiple on/off cycles (Fig. 4 d-e); the photoswitching efficacy was 24.3 ± 3.3%, showing no significant difference from the 33.1 ± 4.3% compared to HEK293T cells expressing Na V 1.5 channels (Fig. 4 h). No significant changes in K + currents were observed under the same experimental conditions (3.0 ± 2.4%), supporting the selectivity of azo-Q2a towards Na V 1.5 channels (Fig. 4 f-h). Additionally, quinidine (in 10 and 30 µM) was applied as control which showed insignificant optical control of both Na + (0.8 ± 0.8%) and K + currents (0.9 ± 1.9%, in 10 µM), (Fig. 4 h and Supplementary Fig. 3a-b). Cryo-EM Structure basis for azo-Q2a optical control of Na V 1.5 channels. To assess azo-Q2a binding we obtained a cryo-EM structure for Na V 1.5 in complex with azo-Q2a at the resolution of 3.1 Å (Na V 1.5S- azo-Q2a ) (Supplementary Table 7). Illuminated by 365 nm light, azo-Q2a was photo-induced as the cis isomer before sample preparation. Note also that, seeking to optimize protein expression, we truncated the carboxyl terminal domain and linker between repeats Ⅰ-Ⅱ and Ⅱ-Ⅲ of Na V 1.5 (Supplementary Fig. 5). We first assessed the inhibition activity of quinidine on Na V 1.5S using whole-cell patch-clamping recording, yielding an IC 50 value of 44.66 µM in the dark and of 43.80 µM under 365 nm illumination; these values do not differ from the values for Na V 1.5 WT (Supplementary Fig. 4a,c and Supplementary Table 5). We then examined the optical control of azo-Q2a towards Na V 1.5S following the same recording protocols and conditions used for Na V 1.5 WT and found that azo-Q2a showed the same inhibition activity and optical control property for Na V 1.5S and Na V 1.5 WT (IC 50 = 10.44 µM for cis ; 75.41 µM for trans , Supplementary Fig. 4b,d), suggesting that truncation of Na V 1.5 does not interfere with the interaction of quinidine or azo-Q2a in the pore domain. The unambiguous EM densities for cis - azo-Q2a are shown in the grey transparent surfaces support that the cis - azo-Q2a molecule bind to the pore domain (Fig. 5 a). Similar to quinidine, cis - azo-Q2a is coordinated by both polar and hydrophobic residues from repeats Ⅰ, Ⅲ, and Ⅳ, but distanced from repeat Ⅱ (Fig. 5 a-b). In addition, upon comparing our complex structure of Na V 1.5S with the reported Na V 1.5-quinidine structure 29 (PDB ID: 6LQA), we found that the binding sites of azo-Q2a and quinidine differ in the central cavity of the pore domain (Fig. 5 c-d): quinidine is likely hydrogen-bonded to Gln371, Thr1417, and Ser1759 and appears to interact with Phe1760 through π-π stacking; cis - azo-Q2a is surrounded by multiple hydrophobic residues from the S6 segments (Fig. 5 d), among which Val405 appears to interact with the quinolone aromatic ring through a carbon-hydrogen··· π interaction (Fig. 5 c), while Phe1760 plays an essential role connecting with the azobenzene group through π-π stacking (Fig. 5 c). The interactions between Phe1760 and the quinoline moiety of quinidine changed to the azobenzene portion of cis - azo-Q2a , while the S6 Ⅳ of Na V 1.5S undergoes an α to π helix transition (Fig. 5 e). We generated Na V 1.5 variants bearing on V405A and F1760A mutations, expressed them in HEK293T cells, and assayed azo-Q2a inhibition activity in the dark or under 365 nm illumination. Assessing the calculated dose-responses supporting our structural insights: the IC 50 of cis isomer azo-Q2a was significantly decreased for the the single V405A mutant variant (IC 50 > 100 µM, approximately 205.1 µM in cis ) compared to the wild-type Na V 1.5 channel (IC 50 = 11.76 µM). Although the specific IC 50 value of azo-Q2a for the single V405A mutant variant were not detected (N.D.) owing to the limited solubility, dose-effect curves showed differences between the dark condition and the 365 nm light (Fig. 5 f-g and Supplementary Table 6). This mutational analysis supports that Val 405 residue contributes to the inhibition activity of azo-Q2a while having little effect on its optical control of Na V 1.5S. Unlike wild-type Na V 1.5 channel, the F1760A mutation did not result in any differential response to azo-Q2a under 365 or 480 nm illumination. The IC 50 of trans - azo-Q2a for blocking Na V 1.5 F1760A in the dark was approximately 173.6 µM, similar to the IC 50 value of 157.3 µM for cis - azo-Q2a under 365 nm illumination (Fig. 5 f,h and Supplementary Table 6). These findings show that the Phe1760 residue is essential for optical control of azo-Q2a towards Na V 1.5S. Together, our Na V 1.5S cis − azo−Q2a and assay data reveal binding sites through which azo-Q2a achieves high inhibition efficiency and optical control towards Na V 1.5S. azo-Q2a reversibly attenuates the heart rate of zebrafish To assess the capability of azo-Q2a to confer optical heart rate control in vivo, we used zebrafish larvae (2 days post-fertilization) as the animal model (Fig. 6 a), for which light scattering is known to be low 30 . The Na + current ( I Na ) generated by Na V 1.5 channels is essential for maintaining a normal heart rate. Heart rate was monitored by counting the number of sequential contractions in 30 s intervals under a dissecting microscope. The larvae were exposed to different treatments (Control; 10 µM azo-Q2a and 100 µM quinidine) for 1-1.5 h in the dark 31 , and then heart rate was recorded 30 min after 365 or 480 nm light exposure using video microscopy (Fig. 6 a). We illuminated control zebrafish larvae that we kept under dissimilar experimental conditions for 5 min (at 365 nm or at 365 nm followed by 480 nm light both continuing for 5 min), aiming to estimate optical effects on the heart rate (Fig. 6 a). We first examined the heart rate of larvae treated with no inhibitor as a control: these showed no significant change after different light exposure, suggesting that the light itself does not affect the heart rate (Fig. 6 b and Supplementary Movie1). The average heart rate of control experiments measured in the dark was used as baseline to normalize each detection value. After perfusion of trans - azo-Q2a (10 µM) lasting for 1-1.5h in the dark, there was no difference in the calculated heart rate (98.8 ± 1.4%) compared to the control treated with DMSO (Supplementary Movie 2). However, we did observe a significant slowing of the heart rate for these larvae after 365nm illumination (56.5 ± 4.3%, Supplementary Movie 3), and this slowing was reversed by the subsequent 480 nm illumination (103.2 ± 2.0%, Fig. 6 b, Supplementary Table 8 and Supplementary Movie 4). We also examined larvae perfused with disposed with quinidine in the dark (90.8 ± 1.4%), under 365 nm illumination (93.9 ± 0.9%) and after subsequent 480 nm illumination (91.6 ± 1.3%), (Fig. 6 b and Supplementary Table 8). These results are consistent with previous reports for quinidine’s inhibition activity, demonstrating that azo-Q2a confers optical control and has higher inhibition of the heart rate in vivo compared with quinidine. Overall, these results indicate that the optical control phenomena of electrophysiological experiments obtained in vitro can be reproduced in living animals and moreover, that azo-Q2a can reversibly control heart rate. Discussion As the first antiarrhythmic drug in the world, quinidine has been used in the treatment of almost all cardiac arrhythmias since the early twentieth century, but in the last two decades it decreases in clinical prescription due to its lack of specificity which leads to intolerable adverse effects in the gastrointestinal tract and nervous system. To date, few teams have chosen to improve the drug properties of quinidine through structural modification. Instead, we here reported on a optopharmacology strategy that not only enables specifically optical control of Na V 1.5 channels for the first time, but enhances its activity and selectivity. The best-performing photoswitchable quinidine derivative modified by azobenzene ( azo-Q2a ) showed 3 fold higher efficacy and only demonstrated optical control of Na V 1.5 function among all our evaluated cardiac ion channels. The high selectivity of it combined with the high spatiotemporal specificity of optopharmacology gives azo-Q2a predictable potential to reduce the adverse effects of quinidine in clinical treatment. We also performed the cryo-EM structure of Na V 1.5S in complex with cis - azo-Q2a , revealing the binding sites through which azo-Q2a achieves high inhibition efficiency and optical control towards Na V 1.5S and providing the chance for further precise modification of azobenzene. light is unsurpassed in its ability to control biological systems with high spatial and temporal resolution. Many Photoswitchable molecules have been reported in recent years such as the photostatins which act as Inhibitors of microtubule dynamics 32 . Our work confirms the feasibility of optical control of natural products and the new use of old drugs through optopharmacology, which not only improves the properties of the drug but also gives it new functions. Notably, we verified the capability of azo-Q2a to confer optical control of heart rate in vivo. On 2 dpf living zebrafish larvae, azo-Q2a enabled optical control of the heart rate, demonstrating its potential in treatment of Na V 1.5 related arrhythmias. Nevertheless, it is incontestable that using the basic azobenzene motif means the optical wavelength of photoswitchable flipping utmost locates near 360 ~ 400 nm and 480 ~ 550 nm 33 . To achieve noninvasive optical control of heart rate in other model animals like mice or even in human require it to have a red shift to nearly 600 nm 34 . Though modification of azobenzene by the ortho halogen or methoxyl substitution can be available to carry out the expectant red shift, our improved azo-Q2a of which the maximum absorbance is in the visible light region, failed to maintain the remarkable optical control of Na V 1.5 channel. Therefore, further modifications of azobenzene motif or other photoswitchable moiety is required. In conclusion, the quinidine-azobenzene ( azo-Q2a ) is a promising photoswitchable molecule targeting Na V 1.5 channel which is worthwhile to future study on cardiac diseases that especially caused by Na V 1.5 mutation. Methods Chemical Synthesis and Photochemical Characterizations Azo -QDs were synthesized and characterized by standard chemical methods. The UV-visible absorption spectra of the cis and trans isomers were determined by UV-Vis spectroscopy. 365 nm and 480 nm LED light was used to perform trans ↔ cis isomerization in vitro. Ratio of the trans and cis isomerization and half-life of cis isomerization was determined by NMR spectra. Full design, synthesis, and photochemical characterization of the Azo -QDs is detailed in the Supplemental Information. Cell culture and transfection The human embryonic kidney cells (HEK293 and HEK-293T) and Chinese hamster ovary cells (CHO) were obtained from ATCC. HEK293 and HEK-293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 15% (volume/volume) Fetal Bovine Serum (FBS, PAN-Biotech) at 37°C and 5% CO 2 . In addition, due to poor adherence of HEK293T stable cell lines expressing K V 4.3 channels and K ir 2.1 channels, 0.1 mg/mL Poly-D-lysine (Sigma) was perfused in culture dishes prior to cell culture and the ratio of FBS increased to 20%. CHO cells were maintained in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (1:1, DMEM/F12, SinoDetech) supplemented with 10% (volume/volume) FBS. Cells with 70 ~ 80% confluence were transiently transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Then the cells were cultured for 20 ~ 48 h and subjected to experiments. Voltage-clamp recordings Voltage-clamp recordings were obtained using a HEKA EPC-10 patch-clamping amplifier (HEKA Electronic) and the data were acquired by the PatchMaster program (HEKA Electronic). For whole-cell patch-clamping recording, patch pipettes were pulled from thin-walled borosilicate glass (Sutter Instrument), polished, and gave resistances of 1.5 to 2.5 MΩ in the experimental solutions. To evaluate the inhibition effect of quinidine and quinidine-azobenzenes , compounds were delivered by gravity perfusion. The illumination during whole-cell patch-clamping recording was given by LumiCite 9100 LED (OPLENIC). For 480 nm wavelength, 100% light intensity was used, and for 365 nm wavelength, 35% light intensity was used. As for the voltage-clamp recording analyses, all data were reported as mean ± SEM. Data analyses were performed using Origin 2022 (Origin Lab), Excel 2016 (Microsoft), and GraphPad Prism 10 (GraphPad Software). Inhibition curves were generated using a Hill equation $$\:\frac{I}{{I}_{max}}=\frac{1}{1+{10}^{(\text{l}\text{o}\text{g}{\text{I}\text{C}}_{50}-[\text{C}\left]\right)\times\:H}}$$ where I is the current at different compounds concentrations, I max is the maximal current of ion channels without inhibitor application, [C] is the logarithmic concentration, IC 50 is the half-maximal inhibition concentration and H is the Hill coefficient. Sodium channel For sodium currents recording, the pipette solution contained 140 mM CsF, 10 mM HEPES, 1 mM EGTA, 10 mM NaCl (pH = 7.3 with CsOH and an osmolarity of ~ 295 mOsm/L). The bath solution contained 140 mM NaCl, 3 mM KCl, 10 mM HEPES, 10 mM Glucose, 1 mM CaCl 2 , 1 mM MgCl 2 (pH = 7.3 with NaOH and an osmolarity of ~ 310 mOsm/L). In whole-cell recordings of Na V 1.5 currents, the cells were held at − 120 mV and the inward peak sodium currents were elicited by a 50 ms step to − 20 mV. In addition, the current-voltage ( I – V ) relationships were obtained by a 50 ms step from − 120 to + 40 mV in 5 mV increment. To calculate the voltage dependence of activation, conductance G was fitting with Boltzmann equation \(\:\frac{G}{{G}_{max}}=\frac{1}{1+{\text{e}}^{\raisebox{1ex}{$({V}_{m}-{V}_{\raisebox{1ex}{$1$}\!\left/\:\!\raisebox{-1ex}{$2$}\right.})$}\!\left/\:\!\raisebox{-1ex}{$-k$}\right.}}\) where G is conductance and G max is the maximum conductance between − 120 and + 40 mV. V m is the stimulus potential. V 1/2 indicates the voltage at half-maximal activation, and k is a slope factor describing voltage sensitivity of the channel. k is the slope factor. G are calculated from the I – V relationships according to \(\:G=\frac{I}{{V}_{m}+{E}_{\text{N}\text{a}}}\) where I is the peak current, G is conductance, V m is the stimulus potential, E Na is the equilibrium potential. Significance of fitted V 1/2 compared to control was analyzed using extra sum-of-squares F test. Potassium channel For potassium currents recording, the pipette solution contained 130 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl 2 , 5 mM Mg-ATP (pH = 7.3 with KOH and an osmolarity of ~ 295 mOsm/L). The bath solution contained 138 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM Glucose, 2 mM CaCl 2 , 1 mM MgCl 2 , 0.33 mM NaH 2 PO 4 ·2H 2 O (pH = 7.3 with NaOH and an osmolarity of ~ 310 mOsm/L). In whole-cell recordings of K V 4.3 currents, the cells were held at − 80 mV then currents were elicited by a 600 ms step to + 60 mV. For hERG currents, the cells were held at − 80 mV then currents were elicited by a step to + 20 mV following a step to -40 mV, each stimulation last for 2 s. For K ir 2.1 currents, the cells were held at − 80 mV then currents were elicited by a 100 ms step to -120 mV. Calcium channel For calcium currents recording, the pipette solution contained 135 mM K-gluconate, 10 mM HEPES, 5 mM EGTA, 2 mM MgCl 2 , 5 mM NaCl, 4 mM Mg-ATP (pH = 7.3 with KOH and an osmolarity of ~ 295 mOsm/L). The bath solution contained 105 mM NaCl, 30 mM TEA-Cl ,10 mM BaCl 2 ·2H 2 O, 10 mM HEPES, 10 mM Glucose, 5 mM CsCl, 4 mM KCl, 1 mM MgCl 2 (pH = 7.3 with NaOH and an osmolarity of ~ 310 mOsm/L). In whole-cell recordings of Ca V 1.2 currents, the cells were held at − 80 mV then currents were elicited by a 200 ms step to -10 mV. Isolate and culture rat ventricular primary cardiomyocytes Ventricular primary cardiomyocytes were isolated from 1- to 2-day-old Sprague-Dawley rats 35 . Briefly, primary cardiomyocytes were isolated with 0.05% trypsin (Gibco) and 0.1% collagenase II (Sigma). Cardiomyocytes were separated from the fibroblasts by pre-plating the digested cell suspension for 2 h. Cells were maintained in DMEM (Gibco) supplemented with 10% (volume/volume) FBS (PAN-Biotech) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Gibco) at 37°C and 5% CO 2 . The voltage-gated whole-cell patch-clamping recording of sodium and potassium currents in primary cardiomyocytes follow the protocol in HEK293T cells. Heart rate recording in zebrafish To determine heart rate of zebrafish, 2 dpf embryos were loaded in a recording chamber filled with E3 solution at the desired stage. Heart rate was calculated by counting the number of sequential contractions in 30 s intervals under a dissecting microscope (S8APO; Leica) 36 . In order to detect the effects of compounds on zebrafish heart rate under different illumination, the control group was without treatment, and the quinidine and azo-Q2a were prepared with E3 buffer at the applicated concentrations. Then, zebrafish larvae were incubated in a 28.5 ℃ incubator in the dark for 1.5 h under perfusion. Next the heart rate labeled darkness was recorded as described above. After that, giving to each group respectively 365 nm light or 365 nm after a subsequent illumination with 480nm light, the duration of each illumination is 5 minutes. Finally, we recorded the heart rate of groups labeled 365 nm or 365 nm + 480 nm after placing the larvae in the dark for 30min after light exposure. Transient co-expression of human Na V 1.5S and β1 The methods for transient co-expression, protein purification, cryo-EM analysis were conducted following a standard protocol 37 . The optimized coding DNAs for human Na V 1.5 (Uniprot: Q14524) was cloned into the pEG BacMam vector with twin Strep-tag and FLAG tag in tandem at the amino terminus, while β1 (Uniprot: Q07699) was cloned into the pCAG vector without affinity tag 38,39 . To optimize the protein behavior, carboxyl terminal domain (1895–2016) and linker between repeat Ⅰ-Ⅱ (461–657) and Ⅱ-Ⅲ (1066–1187) of Na V 1.5, named Na V 15S, were deleted based on the truncated construct rNa V 1.5. HEK293F cells (Invitrogen) were cultured in SMM 293T-I medium (Sino Biological Inc.) under 5% CO 2 in a Multitron-Pro shaker (Infors, 130 r.p.m.) at 37 ℃. When cell density reached 1.8 ~ 2.2 ×10 6 cells/ml, approximately 2.0 mg plasmids (1.5 mg Na V 1.5S and 0.5 mg β1) and 4 mg of 40-kDa linear polyethyleneimines (PEI, Yeasen) were mixed up in 15 mL fresh medium and pre-incubated for 15–30 min before adding into 1 liter cell culture. In addition, 10 mM sodium butyrate was added to cell culture. Transfected cells were cultured for 48 h before harvesting. Protein purification of Na V 1.5 and β1 14 liter transfected cells were harvested by centrifugation at 3800 rpm and resuspended in the lysis buffer containing 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The suspension was supplemented with 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace), 0.1% (w/v) cholesteryl hemisuccinate Tris salt (CHS, Anatrace), and protease inhibitor cocktail containing 2 mM phenylmethylsulfonyl fluoride (PMSF), 6.5 µg/ml aprotinin, 3.5 µg/ml pepstatin, and 25 µg/ml leupeptin. After incubation at 4 ℃ for 2 h, the cell lysate was centrifuged at 13000 rpm for 1 h, and the supernatant was applied to anti-Flag M2 affinity gel (Sigma) at 4°C. After flow through by gravity, the resin was rinsed four times with the Wash buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.02% (w/v) glycol-diosgenin (GDN, Anatrace), and the protease inhibitor cocktail). Protein was eluted with the wash buffer plus 200 µg/mL FLAG peptide (GL.biochem). The eluent was then applied to Strep-Tactin Sepharose (IBA Lifesciences). The purification protocol was similar to the previous steps except for the elution buffer, which was Wash buffer plus 2.5 mM D-Desthiobiotin (IBA Lifesciences). The eluent was then concentrated via 100-kDa cutoff Centricon (Millipore) and further purified with size exclusion chromatography (Superose-6 Increase 10/300 column, GE Healthcare) in Wash buffer. Peak fractions were pooled and concentrated to ∼30 µL. Then, 1 mM quindine-azobenzene (effectiveness 82%) was added to the concentrated sample 30 min before cryo sample preparation. Cryo-EM data acquisition 4 µl aliquots of concentrated Na V 1.5Short- azo-Q2a complex were applied to Quantifoil 300 mesh R1.2/1.3 Au grids which were glow-discharged for 35 s at medium RF level of Plasma Cleaner PDC-32G (Harrick). Then grids were blotted from both sides for 4 s at 8°C and 100% humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Prepared grids were subsequently transferred to a Titan Krios electron microscope (Thermo Fisher) operating at 300 kV and equipped with Cs corrector, Gatan K3 Summit detector and GIF Quantum energy filter. A total of 9,406 movie stacks were automatically collected, using AutoEMation 40 with a slit width of 20 eV on the energy filter and a preset defocus range from − 1.8 µm to -1.5 µm in super-resolution mode at a nominal magnification of 64,000×. Each stack was exposed for 2.56 s with the exposing time of 0.08 s per frame, resulting in total of 32 frames per stack. The total dose rate was 50 e − /Å 2 for each stack. The stacks were motion corrected with MotionCor2 41 and binned 2 fold, resulting in a pixel size of 1.0979 Å/pixel. In addition, dose weighting was performed 42 . The defocus values were estimated with Gctf 43 . Image processing The data processing procedure was nearly identical to the one we previously reported 44 . In brief, a total of 6,525,104 particles were automatically picked in cryoSPARC 45 . 2D classification identified 614,955 good particles that were subsequently applied to Hetero refinement and non-Uniform refinement. Finally, a 3D EM map with an overall resolution of 3.03 Å was generated using 287,376 particles. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion 46 with high resolution noise substitution 47 . Model building and structure refinement The initial model of Na V 1.5S was based on the coordinate of human Na V 1.5-quinidine (PDB:6LQA) 29 , and the model building and structure refinement processes were the same as we used before 44 . In brief, the coordinate of Na V 15-quinidine was fitted into the EM map by CHIMERA 48 . And every residue was manually checked in COOT 49 carefully. The chemical properties of amino acids were considered during model building. The cis - azo-Q2a was fitted into the EM map. Structure refinement was performed using phenix.real_space_refine application in PHENIX 50 real space with secondary structure and geometry restraints. Over-fitting of the overall model was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach and testing the refined model against the other map 51 . Statistics of the map reconstruction and model refinement can be found in Supplementary Table 3. Data analysis and Statistics For in vitro experiments, the cells were evenly suspended and then randomly distributed in each well tested. For in vivo experiments, the zebrafish were distributed into various treatment groups randomly. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software) and SPSS 26.0 software (SPSS Inc.). Before statistical analysis, variation within each group of data and the assumptions of the tests were checked. Comparisons between two independent groups were made using unpaired Student’s two-tailed t test. Comparisons among nonlinear fitted values were made using extra sum-of-squares F test. Comparisons among three or more groups were made using one- or two-way analysis of variance followed by Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Values of IC 50 are presented as mean (95% CI (Profile likelihood)). Other data are presented as mean ± SEM. Declarations Acknowledgements This work was supported by STI2030-Major Projects-2021ZD0202103 (2021ZD0202103 to Z.H.); the National Natural Science Foundation of China (82271498 to Z.H. and 82341246); Ningbo Science and Technology Plan Project (Grant No. 2024Z188); Science Research Project of Hebei Education Department (No.CYZD202501). The authors acknowledge the use of Biorender that is used to create schematic figures. Author Contributions H.Z., H.L., S.L. and W.G. conceived and designed the experiments. S.L. and W.W. carried out the patch-clamping recordings and constructed all the mutations. W.G. synthesized compounds and collected photochemical data. 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2","display":"","copyAsset":false,"role":"figure","size":164916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotochemical characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Photochemical interconversion of \u003cem\u003etrans\u003c/em\u003e- (480 nm illumination, blue) and \u003cem\u003ecis\u003c/em\u003e- (365 nm illumination, violet) \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e, The UV-Vis absorption spectra of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e (100 μM in MeOH:H\u003csub\u003e2\u003c/sub\u003eO = 2:1, rt) in darkness (black, \u003cem\u003etrans\u003c/em\u003e) or at 365 nm (violet, \u003cem\u003ecis\u003c/em\u003e) and 480 nm (blue, \u003cem\u003etrans\u003c/em\u003e) illuminations. \u003cstrong\u003ec\u003c/strong\u003e, \u003cem\u003eA\u003c/em\u003e\u003csub\u003e345\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e440\u003c/sub\u003e of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e plotted over multiple cycles of alternating 365nm (violet) and 480 nm (blue) illuminations (100 μM in MeOH:H\u003csub\u003e2\u003c/sub\u003eO = 2:1, rt). \u003cstrong\u003ed\u003c/strong\u003e, Conversion quantification measured by \u003csup\u003e1\u003c/sup\u003eH-NMR. \u003cstrong\u003ee\u003c/strong\u003e, Thermal relaxation of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e (rt) after converting \u003cem\u003etrans\u003c/em\u003e-\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e to by \u003cem\u003ecis\u003c/em\u003e-\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eillumination with 365 nm light for 5 min.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/19b5c6f6994ac072fb2a82c0.png"},{"id":83547945,"identity":"b0beaabb-5c3e-431c-9dbd-0141ef02d622","added_by":"auto","created_at":"2025-05-28 09:30:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":294544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reversibly inhibits Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e1.5 channels with light in HEK293T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, The Na\u003csub\u003eV\u003c/sub\u003e1.5 inhibition (IC\u003csub\u003e50\u003c/sub\u003e) of quinidine azobenzene derivatives \u003cem\u003e\u003cstrong\u003eazo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Q1-3\u003c/strong\u003e either in darkness (gray, \u003cem\u003etrans\u003c/em\u003e) or at 365 nm (violet, \u003cem\u003ecis\u003c/em\u003e) illumination. Error bars represent 95% Confidence Interval (CI). \u003cstrong\u003eb-h\u003c/strong\u003e, Whole-cell patch clamp recordings of Na\u003csub\u003eV\u003c/sub\u003e1.5 currents through the application of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e and quinidine in darkness (black) or at 480 nm (blue) and 365 nm (violet) illumination. \u003cstrong\u003eb\u003c/strong\u003e, The schematic of experiment design (left) and representative current traces of Na\u003csub\u003eV\u003c/sub\u003e1.5 blocked by varied concentrations of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e and quinidine (right). 0 μM, 10 μM and 100 μM in black, azure and orange respectively. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e, Dose-dependent response curve of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e (\u003cstrong\u003ec\u003c/strong\u003e; n = 9,12) and quinidine (\u003cstrong\u003ed\u003c/strong\u003e; n = 8, 8) in the dark or at 365 nm light. \u003cstrong\u003ee\u003c/strong\u003e, Representative current traces and a diagram of recording protocol of Na\u003csub\u003eV\u003c/sub\u003e1.5 activation (left). \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eI\u003c/em\u003e–\u003cem\u003eV\u003c/em\u003e (\u003cstrong\u003ee\u003c/strong\u003e) and \u003cem\u003eg\u003c/em\u003e–\u003cem\u003eV \u003c/em\u003e(\u003cstrong\u003ef\u003c/strong\u003e) relationship in 10 µM \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eunder 365 nm (n = 15) or 480 nm (n = 12) illumination and without \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e (control, n = 49). Statistical evaluation by ordinary one-way ANOVA. **P \u0026lt; 0.01, ns, not significant (P \u0026gt; 0.05). \u003cstrong\u003eg\u003c/strong\u003e, Representative current traces of reversible photoswitch of Na\u003csub\u003eV\u003c/sub\u003e1.5 by 10 µM \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e. Selected are the last triggered current responses under 365 nm and 480 nm light cycles, each following the protocol showed on \u003cstrong\u003eb\u003c/strong\u003e at 1 Hz for 15 times. Parallel lines represent time gaps between steps. \u003cstrong\u003eh\u003c/strong\u003e, Peak Na\u003csub\u003eV\u003c/sub\u003e1.5 currents of \u003cstrong\u003eg \u003c/strong\u003eover multiple cycles. \u003cstrong\u003ec-f\u003c/strong\u003e, Error bars represent mean ± SEM.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/60ded52e3e9f7ff3b9986119.png"},{"id":83547946,"identity":"b2f20cb5-7c4f-4b97-a4b2-b529a714e574","added_by":"auto","created_at":"2025-05-28 09:30:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":295911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e selectively optical controls Na\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e1.5 channels in cardiomyocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,b\u003c/strong\u003e, Whole-cell patch clamp recordings of Na\u003csub\u003eV\u003c/sub\u003e1.5, K\u003csub\u003eV\u003c/sub\u003e4.3, K\u003csub\u003eir\u003c/sub\u003e2.1, hERG and Ca\u003csub\u003eV\u003c/sub\u003e1.2 currents through application of 10 µM \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e and quinidine at 480 nm (blue), 365 nm (violet) illumination with no inhibition (control, black). \u003cstrong\u003ea\u003c/strong\u003e, Representative current traces and diagrams of recording protocols. \u003cstrong\u003eb\u003c/strong\u003e, Normalized inhibition rate. n = 6-14. \u003cstrong\u003ec\u003c/strong\u003e, Schematic of primary cardiomyocytes patch experiment. \u003cstrong\u003ed\u003c/strong\u003e, Representative current traces of reversible photoswitch of Na\u003csup\u003e+\u003c/sup\u003e current in primary ventricular myocytes induced by 10 µM \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e. Selected are the last triggered current responses under 365 nm and 480 nm light cycles, each following the protocol showed on a (for Na\u003csub\u003eV\u003c/sub\u003e1.5) at 1 Hz for 15 times. e, Peak Na\u003csup\u003e+\u003c/sup\u003e currents of d over multiple cycles. \u003cstrong\u003ed-f\u003c/strong\u003e, Recordings of Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e currents with the same treatment of \u003cstrong\u003ea-b\u003c/strong\u003e. \u003cstrong\u003ef\u003c/strong\u003e. Representative current traces and diagrams of recording protocols. \u003cstrong\u003eg\u003c/strong\u003e, Normalized inhibition rate. n = 5-10. \u003cstrong\u003eh\u003c/strong\u003e, Quantification of photoswitch induced by 10 µM \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e. n = 4-9. Data are statistically compared with Na\u003csub\u003eV\u003c/sub\u003e1.5 group. \u003cstrong\u003ea-h\u003c/strong\u003e, Statistical evaluation by unpaired t test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, not significant (P \u0026gt; 0.05). Error bars represent mean ± SEM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/11578007ead3e8153480dabe.png"},{"id":83548187,"identity":"7b820a53-e5e0-48f5-843f-d4edff07d509","added_by":"auto","created_at":"2025-05-28 09:38:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5040417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular basis for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e coordination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,b\u003c/strong\u003e, \u003cstrong\u003ea\u003c/strong\u003e, \u003cem\u003eCis\u003c/em\u003e-\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e is positioned beneath the ion selectivity filter. Shown here are two perpendicular views Na\u003csub\u003eV\u003c/sub\u003e1.5S-\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e. The four repeats of Na\u003csub\u003eV\u003c/sub\u003e1.5S are colored grey, green, yellow and light blue, respectively. The Ⅲ-Ⅳ linker is colored orange,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e is colored brown, and the sodium ion is colored purple. \u003cstrong\u003eb\u003c/strong\u003e, Local densities for the bound \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e and sodium ion. The densities, shown as grey transparent surfaces, are contoured at 3 σ in ChimeraX. \u003cstrong\u003ec\u003c/strong\u003e, Coordination of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e by residues from repeats Ⅰ, Ⅲ, and Ⅳ. Potential hydrogen bonds are indicated by red dashed lines. \u003cstrong\u003ed\u003c/strong\u003e, Structural comparison of Na\u003csub\u003eV\u003c/sub\u003e1.5S-\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eand Na\u003csub\u003eV\u003c/sub\u003e1.5-quinidine. The structures of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ebound Na\u003csub\u003eV\u003c/sub\u003e1.5S (domain-colored) and quinidine bound Na\u003csub\u003eV\u003c/sub\u003e1.5 (plum, pdb code: 6LQA) are nearly identical. The root-mean-square deviation between them is 0.716 Å over 975 Cα atoms. The binding pose of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e and quinidine is different in the central cavity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Structural differences between Na\u003csub\u003eV\u003c/sub\u003e1.5S-\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e and Na\u003csub\u003eV\u003c/sub\u003e1.5-quinidine. Besides the binding pose of \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e and quinidine, the S6\u003csub\u003eⅣ\u003c/sub\u003e of Na\u003csub\u003eV\u003c/sub\u003e1.5S undergoes an α to π helix transition. \u003cstrong\u003ef\u003c/strong\u003e, The schematic of representative current traces of Na\u003csub\u003eV\u003c/sub\u003e1.5 mutations V405A (left) and F1760A (right) blocked by varied concentrations of\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e. 0 μM, 10 μM and 100 μM in black, azure and orange respectively. \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e, Dose-dependent response curve of V405A (\u003cstrong\u003ed\u003c/strong\u003e; n = 7,6), F1760A (\u003cstrong\u003eh\u003c/strong\u003e; n = 6, 6) by \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e in the dark (black) or at 365 nm (violet) light. Error bars represent mean ± SEM.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/fd35b3a958e1d2b92c341dd0.png"},{"id":83547103,"identity":"ee91fc7b-5b15-4f4e-a6d8-41c018a6e81b","added_by":"auto","created_at":"2025-05-28 09:22:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e optical regulates heart rate in zebrafish.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, The schematic of recording heart rate. \u003cstrong\u003eb\u003c/strong\u003e, The heart rhythm of zebrafish larvae in dark (gray), after 365nm illumination for 5 min (violet) or 365 nm following 480 nm illumination for both 5 min (blue) with no application (control, n = 16-22), 10 µM quinidine (n = 21-22) or 10 µM \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e (n = 11-25). All data are normalized to mean of the control experimental group recording in dark (160 bpm). Statistical evaluation by ordinary one-way ANOVA. ***P \u0026lt; 0.001, ns, not significant (P \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/9bed5954671ebbbd261d5552.png"},{"id":107603873,"identity":"b0bd623b-edf3-4962-bc67-b8a369d94dec","added_by":"auto","created_at":"2026-04-23 07:11:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6562309,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/e41a474a-b422-4a96-8ffb-62183ea8a268.pdf"},{"id":83547095,"identity":"5e798fe3-56d4-4a78-b484-7d68178854ac","added_by":"auto","created_at":"2025-05-28 09:22:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1578360,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/0332577874828446a1cb77f3.docx"},{"id":83547943,"identity":"3a262448-93cd-4ecf-acac-99a4ea186dbb","added_by":"auto","created_at":"2025-05-28 09:30:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":116663,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6532136/v1/54fc7b0bc27688021b322e97.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Optical Control of the Cardiac Rhythm with Photoswitchable NaV1.5 Channel Blockers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Na\u003csub\u003eV\u003c/sub\u003e1.5 encoded by the \u003cem\u003eSCN5A\u003c/em\u003e gene is the predominant subtype of voltage-gated sodium channels family (Na\u003csub\u003eV\u003c/sub\u003e1.1-1.9) in the heart, which plays an essential role in the excitability of cardiomyocytes\u003csup\u003e1\u003c/sup\u003e. The channels mediate the sodium influx into cells resulting in inward sodium current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eNa\u003c/sub\u003e), which induces fast depolarization of rapid response cardiac action potential (AP). Therefore, dysfunction of Na\u003csub\u003eV\u003c/sub\u003e1.5 channels underlies multiple cardiac channelopathies\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e, including long QT syndrome type 3 (LQT3), Brugada syndrome (BrS), sick sinus syndrome (SSS), dilated cardiomyopathy (DCM), atrial fibrillation (AF), and progressive cardiac conduction disease (PCCD). This underscores its status as a pivotal therapeutic target for rhythm management.\u003c/p\u003e \u003cp\u003eAdditionally, given its pivotal role in action potential generation and conduction, Na\u003csub\u003eV\u003c/sub\u003e1.5 has long been recognized as a canonical pharmacological target for Class I antiarrhythmic drugs. Among them, quinidine belongs to the Ⅰ\u003csub\u003ea\u003c/sub\u003e subclass as the first antiarrhythmic drug in the world. Acting as a Na\u003csub\u003eV\u003c/sub\u003e1.5 channel open state blocker, quinidine inhibits peak \u003cem\u003eI\u003c/em\u003e\u003csub\u003eNa\u003c/sub\u003e, thereby reducing the membrane excitability of cardiomyocytes, slowing down the depolarization and conduction velocity of rapid response cardiac AP. It has been previously used for conditions such as atrial premature beats (APB), paroxysmal supraventricular tachycardia (PSVT), AF and LQT3\u003csup\u003e5\u003c/sup\u003e. However, its therapeutic utility is severely constrained by extensive off-target polypharmacology. Quinidine often accompanies inhibition of hERG channel, thereby prolonging APD, ERP and QT interval\u003csup\u003e6,7\u003c/sup\u003e, which increases its off-target activities\u003csup\u003e8\u003c/sup\u003e. Furthermore, quinidine exhibits promiscuous binding to multiple extracardiac receptors, including nicotinic acetylcholine receptors (nAChRs)\u003csup\u003e9\u003c/sup\u003e, 5-hydroxytryptamine receptors 3 (5-HT\u003csub\u003e3\u003c/sub\u003e)\u003csup\u003e10\u003c/sup\u003e and α adrenergic receptors\u003csup\u003e11\u003c/sup\u003e. Its nonspecific pharmacodynamic profile induces intolerable gastrointestinal (nausea, diarrhea) and neurological (tinnitus, cinchonism) adverse effects\u003csup\u003e12\u003c/sup\u003e in approximately 20\u0026ndash;30 percent\u003csup\u003e5\u003c/sup\u003e of patients during clinical application. These limitations underscore the imperative for developing targeted Na\u003csub\u003eV\u003c/sub\u003e1.5 modulators with improved specificity.\u003c/p\u003e \u003cp\u003eLight has emerged as a powerful biophysical modality for spatiotemporally precise control of biological systems\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e. Through its integration with pharmacology, optopharmacology enables non-invasive and rapidly reversible modulation of target-specific receptors and ion channels. This innovative strategy effectively circumvents the problems of off-target and dose-limiting adverse effects of traditional drugs, and thus achieve a much more precise treatment. Moreover, optopharmacology has been widely applied in ion channels\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e, transporters\u003csup\u003e19\u003c/sup\u003e, G protein-coupled receptors\u003csup\u003e20,21\u003c/sup\u003e, receptor-linked enzymes\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHerein, we describe a photoactivated quinidine derivative with a photoswitchable azobenzene moiety incorporating, which enables reversible optical control over Na\u003csub\u003eV\u003c/sub\u003e1.5 function and heart rate. We further characterize the binding sites of our lead compound \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e using single-particle electron cryo-microscopy (cryo-EM). This provides a structural template for achieving selectivity and facilitate rational photoswitchable drug design.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRational design and synthesis of photoswitchable quinidine derivatives\u003c/h2\u003e \u003cp\u003eAs disclosed in the recently cryo-structure of Na\u003csub\u003eV\u003c/sub\u003e1.5 ion channel in complex with quinidine\u003csup\u003e23\u003c/sup\u003e, quinidine blocks the Na\u003csub\u003eV\u003c/sub\u003e1.5 channel mainly with the quinoline moiety. While the quinuclidine group directly interrupts the central permeation pathway, the quinolone aromatic ring primarily interacts with the F1760 residue through a π-π stacking interaction. Much inspired by the above-mentioned crucial interaction, we hypothesized that quinidine derivatives with additional aryl substituents attached to the quinolone core would be beneficial for the binding affinity of Na\u003csub\u003eV\u003c/sub\u003e1.5 ion channel.\u003c/p\u003e \u003cp\u003eInitially, we designed three simple quinidine phenyl derivatives with phenyl substituents at the C-2 (\u003cb\u003eQ1\u003c/b\u003e), C-3 (\u003cb\u003eQ2\u003c/b\u003e), and C-6 (\u003cb\u003eQ3\u003c/b\u003e) positions of the quinoline core (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Specifically, \u003cb\u003eQ1\u003c/b\u003e was synthesized from quinidine via a Minisci reaction\u003csup\u003e24\u003c/sup\u003e while \u003cb\u003eQ2\u003c/b\u003e and \u003cb\u003eQ3\u003c/b\u003e were prepared through Suzuki-Miyaura coupling reactions with phenylboronic acid using \u003cb\u003e3-I-QD\u003c/b\u003e and \u003cb\u003e6-OTf-QD\u003c/b\u003e as starting materials, respectively\u003csup\u003e25,26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRemarkably, whole-cell patch-clamp recordings in HEK293T cells heterologously expressing Na\u003csub\u003eV\u003c/sub\u003e1.5 channels revealed that \u003cb\u003eQ1\u003c/b\u003e and \u003cb\u003eQ3\u003c/b\u003e exhibited significantly higher inhibition rates than quinidine at 10 \u0026micro;M concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). These results strongly suggest that enhanced π-π stacking interactions may strengthen the binding between quinidine derivatives and the Na\u003csub\u003eV\u003c/sub\u003e1.5 ion channel.\u003c/p\u003e \u003cp\u003eNext, we explored photoreactive quinidine derivatives, focusing on azobenzene-based chromophores due to their widespread application. In total, nine azo-quinidine derivatives (\u003cb\u003eazo-QD\u003c/b\u003es) were designed, featuring azobenzene moieties installed at ortho-, meta-, and para-positions with phenyl spacers at the C-2, C-3, and C-6 positions of the quinoline core (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eNotably, our synthetic approach for \u003cb\u003eazo-QD\u003c/b\u003es utilized Suzuki-Miyaura coupling with three key intermediates: \u003cb\u003e3-I-QD\u003c/b\u003e, \u003cb\u003e6-OTf-QD\u003c/b\u003e, and \u003cb\u003e2-Br-QD\u003c/b\u003e (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The synthesis of \u003cb\u003e2-Br-QD\u003c/b\u003e began with the oxidation of quinidine using \u003cem\u003em\u003c/em\u003e-CPBA, followed by reduction with NaHSO\u003csub\u003e3\u003c/sub\u003e, yielding \u003cem\u003eN\u003c/em\u003e-oxide QD in 45% over two steps\u003csup\u003e27\u003c/sup\u003e. Subsequent bromination via the Yu protocol afforded \u003cb\u003e2-Br-QD\u003c/b\u003e in 46% yield\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor \u003cb\u003e3-I-QD\u003c/b\u003e, direct derivatization was achieved through Suzuki-Miyaura cross-coupling with \u003cem\u003ep\u003c/em\u003e-azo-BPin and \u003cem\u003em\u003c/em\u003e-azo-BPin, producing the desired products in 27% and 24% yields, respectively. Meanwhile, \u003cb\u003e6-OTf-QD\u003c/b\u003e and \u003cb\u003e2-Br-QD\u003c/b\u003e were successfully coupled to furnish target compounds in 86%, 48%, 38%, and 33% yields. However, attempts to employ sterically hindered \u003cem\u003eo\u003c/em\u003e-azo-BPin in the Suzuki-Miyaura reaction failed to yield any quinidine derivatives.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhotochemical characterization of quinidine azobenzene derivatives\u003c/h3\u003e\n\u003cp\u003eWith all 6 quinidine azobenzene derivatives in hand, photochemical characterization has been explored (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;1). Using UV-Vis spectroscopy, we characterized the spectral properties of all \u003cem\u003ecis\u003c/em\u003e- and \u003cem\u003etrans\u003c/em\u003e- quinidine azobenzene derivatives (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;1). In the dark, they existed as the thermodynamically favored \u003cem\u003etrans\u003c/em\u003e isomer. Isomerization to the \u003cem\u003ecis\u003c/em\u003e isomer was triggered upon UV-A irradiation (365 nm), and this process was reversed within 5 seconds upon exposure to blue light (480 nm).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken \u003cb\u003eazo-Q2a\u003c/b\u003e as an example, we observed a rapid and reversable photoswitch upon oscillating between 365 and 480 nm illumination over cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Using \u003csup\u003e1\u003c/sup\u003eH-NMR spectroscopy, we assessed the relative concentrations of the two isomers in equilibrium after 365 and 480 nm illumination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). After illumination at 365nm, \u003cem\u003etrans\u003c/em\u003e isomer \u003cb\u003eazo-Q2a\u003c/b\u003e was efficiently isomerized to \u003cem\u003ecis\u003c/em\u003e isomer (86%); 480 nm illumination resulted in efficient isomerization back to the \u003cem\u003etrans\u003c/em\u003e isomer (79%). Finally, we monitored thermal relaxation of the \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e in aqueous solution at room temperature: the half-life for relaxation from the \u003cem\u003ecis\u003c/em\u003e to the \u003cem\u003etrans\u003c/em\u003e isomer was 107h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e\n\u003ch3\u003eOptical control of Na1.5 channels with photoswitchable quinidine azobenzene derivatives\u003c/h3\u003e\n\u003cp\u003eTo determine whether quinidine azobenzene derivatives support optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5 channels, we initially profiled the dose-dependent inhibition activities of quinidine and six derivatives to Na\u003csub\u003eV\u003c/sub\u003e1.5 current in the dark or under 365nm illumination, using whole-cell patch-clamping recordings in HEK293T cells heterologously expressing Na\u003csub\u003eV\u003c/sub\u003e1.5 channels. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d and Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen holding the cellular membrane potential at -120 mV, the expected Na\u003csup\u003e+\u003c/sup\u003e currents were observed at the typical voltage steps\u003csup\u003e29\u003c/sup\u003e (-120 to -20 mV). All of the examined quinidine azobenzene derivatives in the \u003cem\u003ecis\u003c/em\u003e isomer conferred stronger inhibition activity than quinidine, and \u003cb\u003eazo\u003c/b\u003e\u003cb\u003e-\u003c/b\u003e\u003cb\u003e2P\u003c/b\u003e, \u003cb\u003eazo-Q2a\u003c/b\u003e, and \u003cb\u003eazo\u003c/b\u003e\u003cb\u003e-\u003c/b\u003e\u003cb\u003e6M\u003c/b\u003e displayed a significant increase in their IC\u003csub\u003e50\u003c/sub\u003e values after 365 nm illumination. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c and Supplementary Fig.\u0026nbsp;2). Among the examined quinidine azobenzene derivatives, \u003cb\u003eazo-Q2a\u003c/b\u003e showed the most prominent alteration (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.76 \u0026micro;M in \u003cem\u003ecis\u003c/em\u003e; 81.85 \u0026micro;M in \u003cem\u003etrans\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), while quinidine had no obvious difference (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;35.87 \u0026micro;M under 365 nm; 41.21 \u0026micro;M in dark, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). IC\u003csub\u003e50\u003c/sub\u003e values are detailed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eWe measured current-voltage (\u003cem\u003eI\u003c/em\u003e-\u003cem\u003eV\u003c/em\u003e) steps between \u0026minus;\u0026thinsp;120 and +\u0026thinsp;40 mV (5 mV steps) in the presence of 10 \u0026micro;M \u003cb\u003eazo-Q2a\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). In its \u003cem\u003ecis\u003c/em\u003e isomer, \u003cb\u003eazo-Q2a\u003c/b\u003e strongly suppressed inward currents conducted by Na\u003csub\u003eV\u003c/sub\u003e1.5 channels, whereas the \u003cem\u003etrans\u003c/em\u003e isomer had no discernable effect. In addition, neither isomer altered the activation property of Na\u003csub\u003eV\u003c/sub\u003e1.5, assessed as conductance-voltage (\u003cem\u003eg\u003c/em\u003e-\u003cem\u003eV\u003c/em\u003e) relationships and the V\u003csub\u003e50\u003c/sub\u003e value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, Supplementary Table\u0026nbsp;2). Note that blockade of Na\u003csub\u003eV\u003c/sub\u003e1.5 channels was readily reversible as we repeatedly photoswitched \u003cb\u003eazo-Q2a\u003c/b\u003e between its \u003cem\u003etrans\u003c/em\u003e and \u003cem\u003ecis\u003c/em\u003e isomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-h).\u003c/p\u003e \u003cp\u003e \u003cb\u003eazo-Q2a\u003c/b\u003e \u003cb\u003eis highly selective and significantly reduces hERG toxicity of quinidine\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRecalling that quinidine\u0026rsquo;s off-target interactions that inhibit hERG channels lead to serious cardiotoxicity\u003csup\u003e7\u003c/sup\u003e, we investigated the potential inhibition activity of \u003cb\u003eazo-Q2a\u003c/b\u003e towards other ion channels known to contribute to cardiac action potential, including K\u003csub\u003eV\u003c/sub\u003e4.3 (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eto\u003c/sub\u003e), K\u003csub\u003eir\u003c/sub\u003e2.1 (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eK1\u003c/sub\u003e), hERG (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eKr\u003c/sub\u003e) and Ca\u003csub\u003eV\u003c/sub\u003e1.2 (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eCaL\u003c/sub\u003e) channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The experiments monitored the currents of these channels under both 365 and 480 nm illumination. Currents of these ion channels were recorded using whole-cell patch-clamping in HEK293T and CHO cell lines expressing cardiac ion channels (see Methods section for details).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe inhibition rate of 10 \u0026micro;M \u003cb\u003eazo-Q2a\u003c/b\u003e for Na\u003csub\u003eV\u003c/sub\u003e1.5 channels was significantly different between the \u003cem\u003ecis\u003c/em\u003e (61.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8%) and \u003cem\u003etrans\u003c/em\u003e (2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6%) isomers, whereas both \u003cb\u003eazo-Q2a\u003c/b\u003e isomers exerted in same inhibition activity against K\u003csub\u003eV\u003c/sub\u003e4.3, K\u003csub\u003eir\u003c/sub\u003e2.1, hERG and Ca\u003csub\u003eV\u003c/sub\u003e1.2 channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Quantification of the inhibition showed that \u003cb\u003eazo-Q2a\u003c/b\u003e endows light sensitivity only to Na\u003csub\u003eV\u003c/sub\u003e1.5 but has negligible effects on K\u003csub\u003eV\u003c/sub\u003e and Ca\u003csub\u003eV\u003c/sub\u003e channels, yielding photoswitching efficacies of 33.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3% for Na\u003csub\u003eV\u003c/sub\u003e1.5 and far less for the rest channels, of 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% (K\u003csub\u003eV\u003c/sub\u003e4.3), 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2% (K\u003csub\u003eir\u003c/sub\u003e2.1), 7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5% (hERG), 7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4% (Ca\u003csub\u003eV\u003c/sub\u003e1.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh and Supplementary Table\u0026nbsp;4). To compare the difference in hERG channel inhibition activity between \u003cb\u003eazo-Q2a\u003c/b\u003e and quinidine, we repeated these assessments through application of 10 and 30 \u0026micro;M quinidine. As expected, quinidine showed comparable pharmacological behavior across all tested ion channels without optical control property (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;3a-c). Notably, at the same concentration (10 \u0026micro;M), \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e exhibited approximately 7-fold higher Na\u003csub\u003eV\u003c/sub\u003e1.5 inhibition compared to quinidine, while its hERG inhibition activity was roughly half that of quinidine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and Supplementary Table\u0026nbsp;3). In addition, we further evaluated the dose-response relationship of \u003cb\u003eazo-Q2a\u003c/b\u003e and quinidine on hERG channels under 365 and 480 nm illumination, which showed that the IC\u003csub\u003e50\u003c/sub\u003e value of \u003cb\u003eazo-Q2a\u003c/b\u003e on hERG was 16-fold lower than that of quinidine (Supplementary Fig.\u0026nbsp;3d).\u003c/p\u003e \u003cp\u003eTogether, these results demonstrate that \u003cb\u003eazo-Q2a\u003c/b\u003e possesses high selectivity for Na\u003csub\u003eV\u003c/sub\u003e1.5 channels among cardiac ion channels, significantly reducing the hERG channel inhibition activity observed with quinidine. This characteristic makes \u003cb\u003eazo-Q2a\u003c/b\u003e a promising candidate for the development of photoactivated antiarrhythmic therapies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eazo-Q2a\u003c/b\u003e \u003cb\u003ereversibly inhibits Na\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ecurrents in rat primary cardiomyocytes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next investigated \u003cb\u003eazo-Q2a\u003c/b\u003e as an agent for the selective optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5 channels in rat primary cardiomyocytes. We recorded voltage-gated Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e currents from primary ventricular myocytes under 365 and 480 nm illumination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eConsistent with our observations in the transient transfection cell experiments, Na\u003csup\u003e+\u003c/sup\u003e current in cardiomyocytes was reduced at 365 nm illumination after the addition of \u003cb\u003eazo-Q2a\u003c/b\u003e (10 \u0026micro;M), and this was reversed by blue light (480 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The selective optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5 channels was over multiple on/off cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e); the photoswitching efficacy was 24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%, showing no significant difference from the 33.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3% compared to HEK293T cells expressing Na\u003csub\u003eV\u003c/sub\u003e1.5 channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). No significant changes in K\u003csup\u003e+\u003c/sup\u003e currents were observed under the same experimental conditions (3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4%), supporting the selectivity of \u003cb\u003eazo-Q2a\u003c/b\u003e towards Na\u003csub\u003eV\u003c/sub\u003e1.5 channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-h). Additionally, quinidine (in 10 and 30 \u0026micro;M) was applied as control which showed insignificant optical control of both Na\u003csup\u003e+\u003c/sup\u003e (0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%) and K\u003csup\u003e+\u003c/sup\u003e currents (0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9%, in 10 \u0026micro;M), (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh and Supplementary Fig.\u0026nbsp;3a-b).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCryo-EM Structure basis for\u003c/b\u003e \u003cb\u003eazo-Q2a\u003c/b\u003e \u003cb\u003eoptical control of Na\u003c/b\u003e\u003csub\u003e\u003cb\u003eV\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e1.5 channels.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess \u003cb\u003eazo-Q2a\u003c/b\u003e binding we obtained a cryo-EM structure for Na\u003csub\u003eV\u003c/sub\u003e1.5 in complex with \u003cb\u003eazo-Q2a\u003c/b\u003e at the resolution of 3.1 \u0026Aring; (Na\u003csub\u003eV\u003c/sub\u003e1.5S-\u003cb\u003eazo-Q2a\u003c/b\u003e) (Supplementary Table\u0026nbsp;7). Illuminated by 365 nm light, \u003cb\u003eazo-Q2a\u003c/b\u003e was photo-induced as the \u003cem\u003ecis\u003c/em\u003e isomer before sample preparation. Note also that, seeking to optimize protein expression, we truncated the carboxyl terminal domain and linker between repeats Ⅰ-Ⅱ and Ⅱ-Ⅲ of Na\u003csub\u003eV\u003c/sub\u003e1.5 (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003eWe first assessed the inhibition activity of quinidine on Na\u003csub\u003eV\u003c/sub\u003e1.5S using whole-cell patch-clamping recording, yielding an IC\u003csub\u003e50\u003c/sub\u003e value of 44.66 \u0026micro;M in the dark and of 43.80 \u0026micro;M under 365 nm illumination; these values do not differ from the values for Na\u003csub\u003eV\u003c/sub\u003e1.5\u003csup\u003eWT\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;4a,c and Supplementary Table\u0026nbsp;5). We then examined the optical control of \u003cb\u003eazo-Q2a\u003c/b\u003e towards Na\u003csub\u003eV\u003c/sub\u003e1.5S following the same recording protocols and conditions used for Na\u003csub\u003eV\u003c/sub\u003e1.5\u003csup\u003eWT\u003c/sup\u003e and found that \u003cb\u003eazo-Q2a\u003c/b\u003e showed the same inhibition activity and optical control property for Na\u003csub\u003eV\u003c/sub\u003e1.5S and Na\u003csub\u003eV\u003c/sub\u003e1.5\u003csup\u003eWT\u003c/sup\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.44 \u0026micro;M for \u003cem\u003ecis\u003c/em\u003e; 75.41 \u0026micro;M for \u003cem\u003etrans\u003c/em\u003e, Supplementary Fig.\u0026nbsp;4b,d), suggesting that truncation of Na\u003csub\u003eV\u003c/sub\u003e1.5 does not interfere with the interaction of quinidine or \u003cb\u003eazo-Q2a\u003c/b\u003e in the pore domain.\u003c/p\u003e \u003cp\u003eThe unambiguous EM densities for \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e are shown in the grey transparent surfaces support that the \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e molecule bind to the pore domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Similar to quinidine, \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e is coordinated by both polar and hydrophobic residues from repeats Ⅰ, Ⅲ, and Ⅳ, but distanced from repeat Ⅱ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). In addition, upon comparing our complex structure of Na\u003csub\u003eV\u003c/sub\u003e1.5S with the reported Na\u003csub\u003eV\u003c/sub\u003e1.5-quinidine structure\u003csup\u003e29\u003c/sup\u003e (PDB ID: 6LQA), we found that the binding sites of \u003cb\u003eazo-Q2a\u003c/b\u003e and quinidine differ in the central cavity of the pore domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d): quinidine is likely hydrogen-bonded to Gln371, Thr1417, and Ser1759 and appears to interact with Phe1760 through π-π stacking; \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e is surrounded by multiple hydrophobic residues from the S6 segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), among which Val405 appears to interact with the quinolone aromatic ring through a carbon-hydrogen\u0026middot;\u0026middot;\u0026middot; π interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), while Phe1760 plays an essential role connecting with the azobenzene group through π-π stacking (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The interactions between Phe1760 and the quinoline moiety of quinidine changed to the azobenzene portion of \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e, while the S6\u003csub\u003eⅣ\u003c/sub\u003e of Na\u003csub\u003eV\u003c/sub\u003e1.5S undergoes an α to π helix transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe generated Na\u003csub\u003eV\u003c/sub\u003e1.5 variants bearing on V405A and F1760A mutations, expressed them in HEK293T cells, and assayed \u003cb\u003eazo-Q2a\u003c/b\u003e inhibition activity in the dark or under 365 nm illumination. Assessing the calculated dose-responses supporting our structural insights: the IC\u003csub\u003e50\u003c/sub\u003e of \u003cem\u003ecis\u003c/em\u003e isomer \u003cb\u003eazo-Q2a\u003c/b\u003e was significantly decreased for the the single V405A mutant variant (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;100 \u0026micro;M, approximately 205.1 \u0026micro;M in \u003cem\u003ecis\u003c/em\u003e) compared to the wild-type Na\u003csub\u003eV\u003c/sub\u003e1.5 channel (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.76 \u0026micro;M). Although the specific IC\u003csub\u003e50\u003c/sub\u003e value of \u003cb\u003eazo-Q2a\u003c/b\u003e for the single V405A mutant variant were not detected (N.D.) owing to the limited solubility, dose-effect curves showed differences between the dark condition and the 365 nm light (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-g and Supplementary Table\u0026nbsp;6). This mutational analysis supports that Val 405 residue contributes to the inhibition activity of \u003cb\u003eazo-Q2a\u003c/b\u003e while having little effect on its optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5S. Unlike wild-type Na\u003csub\u003eV\u003c/sub\u003e1.5 channel, the F1760A mutation did not result in any differential response to \u003cb\u003eazo-Q2a\u003c/b\u003e under 365 or 480 nm illumination. The IC\u003csub\u003e50\u003c/sub\u003e of \u003cem\u003etrans\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e for blocking Na\u003csub\u003eV\u003c/sub\u003e1.5\u003csup\u003eF1760A\u003c/sup\u003e in the dark was approximately 173.6 \u0026micro;M, similar to the IC\u003csub\u003e50\u003c/sub\u003e value of 157.3 \u0026micro;M for \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e under 365 nm illumination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef,h and Supplementary Table\u0026nbsp;6). These findings show that the Phe1760 residue is essential for optical control of \u003cb\u003eazo-Q2a\u003c/b\u003e towards Na\u003csub\u003eV\u003c/sub\u003e1.5S. Together, our Na\u003csub\u003eV\u003c/sub\u003e1.5S\u003csup\u003e\u003cem\u003ecis\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cb\u003eazo\u0026minus;Q2a\u003c/b\u003e\u003c/sup\u003e and assay data reveal binding sites through which \u003cb\u003eazo-Q2a\u003c/b\u003e achieves high inhibition efficiency and optical control towards Na\u003csub\u003eV\u003c/sub\u003e1.5S.\u003c/p\u003e \u003cp\u003e \u003cb\u003eazo-Q2a\u003c/b\u003e \u003cb\u003ereversibly attenuates the heart rate of zebrafish\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the capability of \u003cb\u003eazo-Q2a\u003c/b\u003e to confer optical heart rate control in vivo, we used zebrafish larvae (2 days post-fertilization) as the animal model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), for which light scattering is known to be low\u003csup\u003e30\u003c/sup\u003e. The Na\u003csup\u003e+\u003c/sup\u003e current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eNa\u003c/sub\u003e) generated by Na\u003csub\u003eV\u003c/sub\u003e1.5 channels is essential for maintaining a normal heart rate. Heart rate was monitored by counting the number of sequential contractions in 30 s intervals under a dissecting microscope.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe larvae were exposed to different treatments (Control; 10 \u0026micro;M \u003cb\u003eazo-Q2a\u003c/b\u003e and 100 \u0026micro;M quinidine) for 1-1.5 h in the dark\u003csup\u003e31\u003c/sup\u003e, and then heart rate was recorded 30 min after 365 or 480 nm light exposure using video microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). We illuminated control zebrafish larvae that we kept under dissimilar experimental conditions for 5 min (at 365 nm or at 365 nm followed by 480 nm light both continuing for 5 min), aiming to estimate optical effects on the heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eWe first examined the heart rate of larvae treated with no inhibitor as a control: these showed no significant change after different light exposure, suggesting that the light itself does not affect the heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and Supplementary Movie1). The average heart rate of control experiments measured in the dark was used as baseline to normalize each detection value. After perfusion of \u003cem\u003etrans\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e (10 \u0026micro;M) lasting for 1-1.5h in the dark, there was no difference in the calculated heart rate (98.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%) compared to the control treated with DMSO (Supplementary Movie 2). However, we did observe a significant slowing of the heart rate for these larvae after 365nm illumination (56.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3%, Supplementary Movie 3), and this slowing was reversed by the subsequent 480 nm illumination (103.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0%, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, Supplementary Table\u0026nbsp;8 and Supplementary Movie 4).\u003c/p\u003e \u003cp\u003eWe also examined larvae perfused with disposed with quinidine in the dark (90.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%), under 365 nm illumination (93.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%) and after subsequent 480 nm illumination (91.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%), (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and Supplementary Table\u0026nbsp;8). These results are consistent with previous reports for quinidine\u0026rsquo;s inhibition activity, demonstrating that \u003cb\u003eazo-Q2a\u003c/b\u003e confers optical control and has higher inhibition of the heart rate in vivo compared with quinidine.\u003c/p\u003e \u003cp\u003eOverall, these results indicate that the optical control phenomena of electrophysiological experiments obtained in vitro can be reproduced in living animals and moreover, that \u003cb\u003eazo-Q2a\u003c/b\u003e can reversibly control heart rate.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs the first antiarrhythmic drug in the world, quinidine has been used in the treatment of almost all cardiac arrhythmias since the early twentieth century, but in the last two decades it decreases in clinical prescription due to its lack of specificity which leads to intolerable adverse effects in the gastrointestinal tract and nervous system. To date, few teams have chosen to improve the drug properties of quinidine through structural modification. Instead, we here reported on a optopharmacology strategy that not only enables specifically optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5 channels for the first time, but enhances its activity and selectivity. The best-performing photoswitchable quinidine derivative modified by azobenzene (\u003cb\u003eazo-Q2a\u003c/b\u003e) showed 3 fold higher efficacy and only demonstrated optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5 function among all our evaluated cardiac ion channels. The high selectivity of it combined with the high spatiotemporal specificity of optopharmacology gives \u003cb\u003eazo-Q2a\u003c/b\u003e predictable potential to reduce the adverse effects of quinidine in clinical treatment. We also performed the cryo-EM structure of Na\u003csub\u003eV\u003c/sub\u003e1.5S in complex with \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e, revealing the binding sites through which \u003cb\u003eazo-Q2a\u003c/b\u003e achieves high inhibition efficiency and optical control towards Na\u003csub\u003eV\u003c/sub\u003e1.5S and providing the chance for further precise modification of azobenzene.\u003c/p\u003e \u003cp\u003elight is unsurpassed in its ability to control biological systems with high spatial and temporal resolution. Many Photoswitchable molecules have been reported in recent years such as the photostatins which act as Inhibitors of microtubule dynamics\u003csup\u003e32\u003c/sup\u003e. Our work confirms the feasibility of optical control of natural products and the new use of old drugs through optopharmacology, which not only improves the properties of the drug but also gives it new functions.\u003c/p\u003e \u003cp\u003eNotably, we verified the capability of \u003cb\u003eazo-Q2a\u003c/b\u003e to confer optical control of heart rate in vivo. On 2 dpf living zebrafish larvae, \u003cb\u003eazo-Q2a\u003c/b\u003e enabled optical control of the heart rate, demonstrating its potential in treatment of Na\u003csub\u003eV\u003c/sub\u003e1.5 related arrhythmias. Nevertheless, it is incontestable that using the basic azobenzene motif means the optical wavelength of photoswitchable flipping utmost locates near 360\u0026thinsp;~\u0026thinsp;400 nm and 480\u0026thinsp;~\u0026thinsp;550 nm\u003csup\u003e33\u003c/sup\u003e. To achieve noninvasive optical control of heart rate in other model animals like mice or even in human require it to have a red shift to nearly 600 nm\u003csup\u003e34\u003c/sup\u003e. Though modification of azobenzene by the ortho halogen or methoxyl substitution can be available to carry out the expectant red shift, our improved \u003cb\u003eazo-Q2a\u003c/b\u003e of which the maximum absorbance is in the visible light region, failed to maintain the remarkable optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5 channel. Therefore, further modifications of azobenzene motif or other photoswitchable moiety is required.\u003c/p\u003e \u003cp\u003eIn conclusion, the \u003cb\u003equinidine-azobenzene\u003c/b\u003e (\u003cb\u003eazo-Q2a\u003c/b\u003e) is a promising photoswitchable molecule targeting Na\u003csub\u003eV\u003c/sub\u003e1.5 channel which is worthwhile to future study on cardiac diseases that especially caused by Na\u003csub\u003eV\u003c/sub\u003e1.5 mutation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChemical Synthesis and Photochemical Characterizations\u003c/h2\u003e \u003cp\u003e \u003cb\u003eAzo\u003c/b\u003e \u003cb\u003e-QDs\u003c/b\u003e were synthesized and characterized by standard chemical methods. The UV-visible absorption spectra of the \u003cem\u003ecis\u003c/em\u003e and \u003cem\u003etrans\u003c/em\u003e isomers were determined by UV-Vis spectroscopy. 365 nm and 480 nm LED light was used to perform \u003cem\u003etrans\u003c/em\u003e \u0026harr; \u003cem\u003ecis\u003c/em\u003e isomerization in vitro. Ratio of the \u003cem\u003etrans\u003c/em\u003e and \u003cem\u003ecis\u003c/em\u003e isomerization and half-life of \u003cem\u003ecis\u003c/em\u003e isomerization was determined by NMR spectra. Full design, synthesis, and photochemical characterization of the \u003cb\u003eAzo\u003c/b\u003e\u003cb\u003e-QDs\u003c/b\u003e is detailed in the Supplemental Information.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and transfection\u003c/h3\u003e\n\u003cp\u003eThe human embryonic kidney cells (HEK293 and HEK-293T) and Chinese hamster ovary cells (CHO) were obtained from ATCC. HEK293 and HEK-293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 15% (volume/volume) Fetal Bovine Serum (FBS, PAN-Biotech) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. In addition, due to poor adherence of HEK293T stable cell lines expressing K\u003csub\u003eV\u003c/sub\u003e4.3 channels and K\u003csub\u003eir\u003c/sub\u003e2.1 channels, 0.1 mg/mL Poly-D-lysine (Sigma) was perfused in culture dishes prior to cell culture and the ratio of FBS increased to 20%. CHO cells were maintained in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (1:1, DMEM/F12, SinoDetech) supplemented with 10% (volume/volume) FBS.\u003c/p\u003e \u003cp\u003eCells with 70\u0026thinsp;~\u0026thinsp;80% confluence were transiently transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Then the cells were cultured for 20\u0026thinsp;~\u0026thinsp;48 h and subjected to experiments.\u003c/p\u003e\n\u003ch3\u003eVoltage-clamp recordings\u003c/h3\u003e\n\u003cp\u003eVoltage-clamp recordings were obtained using a HEKA EPC-10 patch-clamping amplifier (HEKA Electronic) and the data were acquired by the PatchMaster program (HEKA Electronic). For whole-cell patch-clamping recording, patch pipettes were pulled from thin-walled borosilicate glass (Sutter Instrument), polished, and gave resistances of 1.5 to 2.5 MΩ in the experimental solutions. To evaluate the inhibition effect of quinidine and \u003cb\u003equinidine-azobenzenes\u003c/b\u003e, compounds were delivered by gravity perfusion. The illumination during whole-cell patch-clamping recording was given by LumiCite 9100 LED (OPLENIC). For 480 nm wavelength, 100% light intensity was used, and for 365 nm wavelength, 35% light intensity was used.\u003c/p\u003e \u003cp\u003eAs for the voltage-clamp recording analyses, all data were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Data analyses were performed using Origin 2022 (Origin Lab), Excel 2016 (Microsoft), and GraphPad Prism 10 (GraphPad Software). Inhibition curves were generated using a Hill equation\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\frac{I}{{I}_{max}}=\\frac{1}{1+{10}^{(\\text{l}\\text{o}\\text{g}{\\text{I}\\text{C}}_{50}-[\\text{C}\\left]\\right)\\times\\:H}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eI\u003c/em\u003e is the current at different compounds concentrations, \u003cem\u003eI\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e is the maximal current of ion channels without inhibitor application, [C] is the logarithmic concentration, IC\u003csub\u003e50\u003c/sub\u003e is the half-maximal inhibition concentration and \u003cem\u003eH\u003c/em\u003e is the Hill coefficient.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSodium channel\u003c/h2\u003e \u003cp\u003eFor sodium currents recording, the pipette solution contained 140 mM CsF, 10 mM HEPES, 1 mM EGTA, 10 mM NaCl (pH\u0026thinsp;=\u0026thinsp;7.3 with CsOH and an osmolarity of ~\u0026thinsp;295 mOsm/L). The bath solution contained 140 mM NaCl, 3 mM KCl, 10 mM HEPES, 10 mM Glucose, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e (pH\u0026thinsp;=\u0026thinsp;7.3 with NaOH and an osmolarity of ~\u0026thinsp;310 mOsm/L). In whole-cell recordings of Na\u003csub\u003eV\u003c/sub\u003e1.5 currents, the cells were held at \u0026minus;\u0026thinsp;120 mV and the inward peak sodium currents were elicited by a 50 ms step to \u0026minus;\u0026thinsp;20 mV. In addition, the current-voltage (\u003cem\u003eI\u003c/em\u003e\u0026ndash;\u003cem\u003eV\u003c/em\u003e) relationships were obtained by a 50 ms step from \u0026minus;\u0026thinsp;120 to +\u0026thinsp;40 mV in 5 mV increment. To calculate the voltage dependence of activation, conductance \u003cem\u003eG\u003c/em\u003e was fitting with Boltzmann equation\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{G}{{G}_{max}}=\\frac{1}{1+{\\text{e}}^{\\raisebox{1ex}{$({V}_{m}-{V}_{\\raisebox{1ex}{$1$}\\!\\left/\\:\\!\\raisebox{-1ex}{$2$}\\right.})$}\\!\\left/\\:\\!\\raisebox{-1ex}{$-k$}\\right.}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eG\u003c/em\u003e is conductance and \u003cem\u003eG\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e is the maximum conductance between \u0026minus;\u0026thinsp;120 and +\u0026thinsp;40 mV. \u003cem\u003eV\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e is the stimulus potential. \u003cem\u003eV\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e indicates the voltage at half-maximal activation, and \u003cem\u003ek\u003c/em\u003e is a slope factor describing voltage sensitivity of the channel. \u003cem\u003ek\u003c/em\u003e is the slope factor.\u003cem\u003eG\u003c/em\u003e are calculated from the \u003cem\u003eI\u003c/em\u003e\u0026ndash;\u003cem\u003eV\u003c/em\u003e relationships according to\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:G=\\frac{I}{{V}_{m}+{E}_{\\text{N}\\text{a}}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eI\u003c/em\u003e is the peak current, \u003cem\u003eG\u003c/em\u003e is conductance, \u003cem\u003eV\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e is the stimulus potential, \u003cem\u003eE\u003c/em\u003e\u003csub\u003eNa\u003c/sub\u003e is the equilibrium potential. Significance of fitted \u003cem\u003eV\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e compared to control was analyzed using extra sum-of-squares F test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePotassium channel\u003c/h2\u003e \u003cp\u003eFor potassium currents recording, the pipette solution contained 130 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM Mg-ATP (pH\u0026thinsp;=\u0026thinsp;7.3 with KOH and an osmolarity of ~\u0026thinsp;295 mOsm/L). The bath solution contained 138 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM Glucose, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.33 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO (pH\u0026thinsp;=\u0026thinsp;7.3 with NaOH and an osmolarity of ~\u0026thinsp;310 mOsm/L). In whole-cell recordings of K\u003csub\u003eV\u003c/sub\u003e4.3 currents, the cells were held at \u0026minus;\u0026thinsp;80 mV then currents were elicited by a 600 ms step to +\u0026thinsp;60 mV. For hERG currents, the cells were held at \u0026minus;\u0026thinsp;80 mV then currents were elicited by a step to +\u0026thinsp;20 mV following a step to -40 mV, each stimulation last for 2 s. For K\u003csub\u003eir\u003c/sub\u003e2.1 currents, the cells were held at \u0026minus;\u0026thinsp;80 mV then currents were elicited by a 100 ms step to -120 mV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCalcium channel\u003c/h2\u003e \u003cp\u003eFor calcium currents recording, the pipette solution contained 135 mM K-gluconate, 10 mM HEPES, 5 mM EGTA, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM NaCl, 4 mM Mg-ATP (pH\u0026thinsp;=\u0026thinsp;7.3 with KOH and an osmolarity of ~\u0026thinsp;295 mOsm/L). The bath solution contained 105 mM NaCl, 30 mM TEA-Cl ,10 mM BaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 10 mM HEPES, 10 mM Glucose, 5 mM CsCl, 4 mM KCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e (pH\u0026thinsp;=\u0026thinsp;7.3 with NaOH and an osmolarity of ~\u0026thinsp;310 mOsm/L). In whole-cell recordings of Ca\u003csub\u003eV\u003c/sub\u003e1.2 currents, the cells were held at \u0026minus;\u0026thinsp;80 mV then currents were elicited by a 200 ms step to -10 mV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIsolate and culture rat ventricular primary cardiomyocytes\u003c/h2\u003e \u003cp\u003eVentricular primary cardiomyocytes were isolated from 1- to 2-day-old Sprague-Dawley rats\u003csup\u003e35\u003c/sup\u003e. Briefly, primary cardiomyocytes were isolated with 0.05% trypsin (Gibco) and 0.1% collagenase II (Sigma). Cardiomyocytes were separated from the fibroblasts by pre-plating the digested cell suspension for 2 h. Cells were maintained in DMEM (Gibco) supplemented with 10% (volume/volume) FBS (PAN-Biotech) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Gibco) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe voltage-gated whole-cell patch-clamping recording of sodium and potassium currents in primary cardiomyocytes follow the protocol in HEK293T cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHeart rate recording in zebrafish\u003c/h2\u003e \u003cp\u003eTo determine heart rate of zebrafish, 2 dpf embryos were loaded in a recording chamber filled with E3 solution at the desired stage. Heart rate was calculated by counting the number of sequential contractions in 30 s intervals under a dissecting microscope (S8APO; Leica)\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn order to detect the effects of compounds on zebrafish heart rate under different illumination, the control group was without treatment, and the quinidine and \u003cb\u003eazo-Q2a\u003c/b\u003e were prepared with E3 buffer at the applicated concentrations. Then, zebrafish larvae were incubated in a 28.5 ℃ incubator in the dark for 1.5 h under perfusion. Next the heart rate labeled darkness was recorded as described above. After that, giving to each group respectively 365 nm light or 365 nm after a subsequent illumination with 480nm light, the duration of each illumination is 5 minutes. Finally, we recorded the heart rate of groups labeled 365 nm or 365 nm\u0026thinsp;+\u0026thinsp;480 nm after placing the larvae in the dark for 30min after light exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTransient co-expression of human Na\u003csub\u003eV\u003c/sub\u003e1.5S and β1\u003c/h2\u003e \u003cp\u003eThe methods for transient co-expression, protein purification, cryo-EM analysis were conducted following a standard protocol\u003csup\u003e37\u003c/sup\u003e. The optimized coding DNAs for human Na\u003csub\u003eV\u003c/sub\u003e1.5 (Uniprot: Q14524) was cloned into the pEG BacMam vector with twin Strep-tag and FLAG tag in tandem at the amino terminus, while β1 (Uniprot: Q07699) was cloned into the pCAG vector without affinity tag\u003csup\u003e38,39\u003c/sup\u003e. To optimize the protein behavior, carboxyl terminal domain (1895\u0026ndash;2016) and linker between repeat Ⅰ-Ⅱ (461\u0026ndash;657) and Ⅱ-Ⅲ (1066\u0026ndash;1187) of Na\u003csub\u003eV\u003c/sub\u003e1.5, named Na\u003csub\u003eV\u003c/sub\u003e15S, were deleted based on the truncated construct rNa\u003csub\u003eV\u003c/sub\u003e1.5. HEK293F cells (Invitrogen) were cultured in SMM 293T-I medium (Sino Biological Inc.) under 5% CO\u003csub\u003e2\u003c/sub\u003e in a Multitron-Pro shaker (Infors, 130 r.p.m.) at 37 ℃. When cell density reached 1.8\u0026thinsp;~\u0026thinsp;2.2 \u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml, approximately 2.0 mg plasmids (1.5 mg Na\u003csub\u003eV\u003c/sub\u003e1.5S and 0.5 mg β1) and 4 mg of 40-kDa linear polyethyleneimines (PEI, Yeasen) were mixed up in 15 mL fresh medium and pre-incubated for 15\u0026ndash;30 min before adding into 1 liter cell culture. In addition, 10 mM sodium butyrate was added to cell culture. Transfected cells were cultured for 48 h before harvesting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProtein purification of Na\u003csub\u003eV\u003c/sub\u003e1.5 and β1\u003c/h2\u003e \u003cp\u003e14 liter transfected cells were harvested by centrifugation at 3800 rpm and resuspended in the lysis buffer containing 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The suspension was supplemented with 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace), 0.1% (w/v) cholesteryl hemisuccinate Tris salt (CHS, Anatrace), and protease inhibitor cocktail containing 2 mM phenylmethylsulfonyl fluoride (PMSF), 6.5 \u0026micro;g/ml aprotinin, 3.5 \u0026micro;g/ml pepstatin, and 25 \u0026micro;g/ml leupeptin. After incubation at 4 ℃ for 2 h, the cell lysate was centrifuged at 13000 rpm for 1 h, and the supernatant was applied to anti-Flag M2 affinity gel (Sigma) at 4\u0026deg;C. After flow through by gravity, the resin was rinsed four times with the Wash buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.02% (w/v) glycol-diosgenin (GDN, Anatrace), and the protease inhibitor cocktail). Protein was eluted with the wash buffer plus 200 \u0026micro;g/mL FLAG peptide (GL.biochem). The eluent was then applied to Strep-Tactin Sepharose (IBA Lifesciences). The purification protocol was similar to the previous steps except for the elution buffer, which was Wash buffer plus 2.5 mM D-Desthiobiotin (IBA Lifesciences). The eluent was then concentrated via 100-kDa cutoff Centricon (Millipore) and further purified with size exclusion chromatography (Superose-6 Increase 10/300 column, GE Healthcare) in Wash buffer. Peak fractions were pooled and concentrated to \u0026sim;30 \u0026micro;L. Then, 1 mM quindine-azobenzene (effectiveness 82%) was added to the concentrated sample 30 min before cryo sample preparation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCryo-EM data acquisition\u003c/h2\u003e \u003cp\u003e4 \u0026micro;l aliquots of concentrated Na\u003csub\u003eV\u003c/sub\u003e1.5Short-\u003cb\u003eazo-Q2a\u003c/b\u003e complex were applied to Quantifoil 300 mesh R1.2/1.3 Au grids which were glow-discharged for 35 s at medium RF level of Plasma Cleaner PDC-32G (Harrick). Then grids were blotted from both sides for 4 s at 8\u0026deg;C and 100% humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Prepared grids were subsequently transferred to a Titan Krios electron microscope (Thermo Fisher) operating at 300 kV and equipped with Cs corrector, Gatan K3 Summit detector and GIF Quantum energy filter. A total of 9,406 movie stacks were automatically collected, using AutoEMation\u003csup\u003e40\u003c/sup\u003e with a slit width of 20 eV on the energy filter and a preset defocus range from \u0026minus;\u0026thinsp;1.8 \u0026micro;m to -1.5 \u0026micro;m in super-resolution mode at a nominal magnification of 64,000\u0026times;. Each stack was exposed for 2.56 s with the exposing time of 0.08 s per frame, resulting in total of 32 frames per stack. The total dose rate was 50 e\u003csup\u003e\u0026minus;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e for each stack. The stacks were motion corrected with MotionCor2\u003csup\u003e41\u003c/sup\u003e and binned 2 fold, resulting in a pixel size of 1.0979 \u0026Aring;/pixel. In addition, dose weighting was performed\u003csup\u003e42\u003c/sup\u003e. The defocus values were estimated with Gctf\u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eImage processing\u003c/h2\u003e \u003cp\u003eThe data processing procedure was nearly identical to the one we previously reported\u003csup\u003e44\u003c/sup\u003e. In brief, a total of 6,525,104 particles were automatically picked in cryoSPARC\u003csup\u003e45\u003c/sup\u003e. 2D classification identified 614,955 good particles that were subsequently applied to Hetero refinement and non-Uniform refinement. Finally, a 3D EM map with an overall resolution of 3.03 \u0026Aring; was generated using 287,376 particles. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion\u003csup\u003e46\u003c/sup\u003e with high resolution noise substitution\u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eModel building and structure refinement\u003c/h2\u003e \u003cp\u003eThe initial model of Na\u003csub\u003eV\u003c/sub\u003e1.5S was based on the coordinate of human Na\u003csub\u003eV\u003c/sub\u003e1.5-quinidine (PDB:6LQA)\u003csup\u003e29\u003c/sup\u003e, and the model building and structure refinement processes were the same as we used before\u003csup\u003e44\u003c/sup\u003e. In brief, the coordinate of Na\u003csub\u003eV\u003c/sub\u003e15-quinidine was fitted into the EM map by CHIMERA\u003csup\u003e48\u003c/sup\u003e. And every residue was manually checked in COOT\u003csup\u003e49\u003c/sup\u003e carefully. The chemical properties of amino acids were considered during model building. The \u003cem\u003ecis\u003c/em\u003e-\u003cb\u003eazo-Q2a\u003c/b\u003e was fitted into the EM map. Structure refinement was performed using phenix.real_space_refine application in PHENIX\u003csup\u003e50\u003c/sup\u003e real space with secondary structure and geometry restraints. Over-fitting of the overall model was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach and testing the refined model against the other map\u003csup\u003e51\u003c/sup\u003e. Statistics of the map reconstruction and model refinement can be found in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData analysis and Statistics\u003c/h2\u003e \u003cp\u003eFor in vitro experiments, the cells were evenly suspended and then randomly distributed in each well tested. For in vivo experiments, the zebrafish were distributed into various treatment groups randomly. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software) and SPSS 26.0 software (SPSS Inc.). Before statistical analysis, variation within each group of data and the assumptions of the tests were checked. Comparisons between two independent groups were made using unpaired Student\u0026rsquo;s two-tailed t test. Comparisons among nonlinear fitted values were made using extra sum-of-squares F test. Comparisons among three or more groups were made using one- or two-way analysis of variance followed by Bonferroni\u0026rsquo;s post hoc test. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Values of IC\u003csub\u003e50\u003c/sub\u003e are presented as mean (95% CI (Profile likelihood)). Other data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was supported by STI2030-Major Projects-2021ZD0202103 (2021ZD0202103 to Z.H.); the National Natural Science Foundation of China (82271498 to Z.H. and 82341246); Ningbo Science and Technology Plan Project (Grant No. 2024Z188); Science Research Project of Hebei Education Department (No.CYZD202501). The authors acknowledge the use of Biorender that is used to create schematic figures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH.Z., H.L., S.L. and W.G. conceived and designed the experiments. S.L. and W.W. carried out the patch-clamping recordings and constructed all the mutations. W.G. synthesized compounds and collected photochemical data. S.L. and J.H. collected heart rate data in zebrafish. Z.L., J.L., and N.Y. performed all the experiments of cryo-EM data. S.L., W.G., Z.L., H.Z. and H.L. wrote the paper. All authors reviewed and revised the paper.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Authors declare no competing interests.\u003c/p\u003e"},{"header":"REFERENCES","content":"\u003col\u003e\n \u003cli\u003eChen, L., He, Y., Wang, X., Ge, J. \u0026amp; Li, H. Ventricular voltage-gated ion channels: Detection, characteristics, mechanisms, and drug safety evaluation. \u003cem\u003eClin Transl Med\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e530 (2021).\u003c/li\u003e\n \u003cli\u003eVeerman, C.C., Wilde, A.A. \u0026amp; Lodder, E.M. The cardiac sodium channel gene SCN5A and its gene product NaV1.5: Role in physiology and pathophysiology. \u003cem\u003eGene\u003c/em\u003e \u003cstrong\u003e573\u003c/strong\u003e, 177-87 (2015).\u003c/li\u003e\n \u003cli\u003eWilde, A.A.M. \u0026amp; Amin, A.S. 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Dysfunction of Na\u003csub\u003eV\u003c/sub\u003e1.5 is implicated in life-threatening arrhythmias, making it a critical therapeutic target. Acting as a Na\u003csub\u003eV\u003c/sub\u003e1.5 open-state blocker, quinidine demonstrates efficacy in arrhythmia treatment, but its low specificity restricts its clinical application. Here, we reported an optopharmacological strategy which enables a precise and optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5 function by means of photoswitchable quinidine derivatives. Through systematic structural optimization, we identified \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e as a high-performance photoswitchable inhibitor, exhibiting low activity in the dark or under 480 nm light irradiation (\u003cem\u003etrans\u003c/em\u003e isomer), while approximately 7-fold higher efficacy was observed under 365 nm light irradiation (\u003cem\u003ecis\u003c/em\u003e isomer). Of note, \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e demonstrated exceptional selectivity for Na\u003csub\u003eV\u003c/sub\u003e1.5 over other cardiac ion channels, minimizing potential off-target effects. Furthermore, by solving the cryo-EM structure of the Na\u003csub\u003eV\u003c/sub\u003e1.5 in complex with the \u003cem\u003ecis\u003c/em\u003e-active isomer \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e (3.1 Å resolution), we revealed the essential binding site that is responsible for the optical control of Na\u003csub\u003eV\u003c/sub\u003e1.5. Finally, \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e also attenuates heart rate of living zebrafish larvae with light, showing its potential in cardiac related research and treatment. Our work not only establishes \u003cem\u003e\u003cstrong\u003eazo-Q2a\u003c/strong\u003e\u003c/em\u003e as a robust photoswitchable inhibitor for Na\u003csub\u003eV\u003c/sub\u003e1.5 but also provides a structural blueprint for the rational design of next-generation optopharmacological antiarrhythmic agents.\u003c/p\u003e","manuscriptTitle":"Optical Control of the Cardiac Rhythm with Photoswitchable NaV1.5 Channel Blockers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-28 09:22:08","doi":"10.21203/rs.3.rs-6532136/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b67a960-8aac-42fc-bbd2-7dc0660f6696","owner":[],"postedDate":"May 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48277129,"name":"Biological sciences/Chemical biology/Ion channels"},{"id":48277130,"name":"Health sciences/Diseases/Cardiovascular diseases"}],"tags":[],"updatedAt":"2026-04-23T07:11:29+00:00","versionOfRecord":{"articleIdentity":"rs-6532136","link":"https://doi.org/10.1038/s41467-026-70305-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-03-10 04:00:00","publishedOnDateReadable":"March 10th, 2026"},"versionCreatedAt":"2025-05-28 09:22:08","video":"","vorDoi":"10.1038/s41467-026-70305-6","vorDoiUrl":"https://doi.org/10.1038/s41467-026-70305-6","workflowStages":[]},"version":"v1","identity":"rs-6532136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6532136","identity":"rs-6532136","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}