Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells

Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells Xinyu Li, Li Zhao, Rongrong Feng, Xiaowei Du, Zelin Guo, Yu Meng, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2480271/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Voltage-gated sodium channels (VGSC) are widely expressed in various types of tumor and cancer cells, and Na V 1.5 are overexpression in highly metastatic breast cancer cells. There may be positive relations between the expression levels of Na V 1.5 and the breast cancer recurrence and metastasis. Herein, Na V 1.5 were detected and localized on the surfaces of normal and cancer breast cells by Single Molecular Recognition Imaging mode (SMRI) of Atomic Force Microscopy (AFM). The results reveal that Na V 1.5 were irregularly distributed on the surfaces of normal and cancer breast cells. The Na V 1.5 take an area percentage of 0.6% and 7.2% on normal and cancer breast cells, respectively, which indicates that there are more Na V 1.5 on cancer cells than normal cells. The specific interaction forces and binding kinetics between Na V 1.5-antibody complex system were investigated with Single Molecular Force Spectroscopy (SMFS) mode of AFM, indicating that the stability of the Na V 1.5-antibody on normal breast cells is higher than cancer breast cells. All these results will be useful to study the interactions of other ion channel-antibody systems, and will also be useful to understand the role of sodium channels in tumor metastasis and invasion. Voltage-gated sodium channels breast cells Atomic Force Microscopy Single Molecular Recognition Imaging Single Molecular Force microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction VGSC are transmembrane proteins that can adjust the movements of sodium ions across cellular membranes along an electrochemical gradient [ 1 ]. Mammalian VGSC are formed by a large pseudo tetrameric pore forming α subunit (220 ~ 260 kDa) and one or two β subunits (33–36 kDa). There are nine different α subunits (Na V 1.1 ~ Na V 1.9) that show tissue specific expression profiles [ 1 , 2 ]. The opening and closing of the sodium channels are regulated by the activation and inactivation gating processes, respectively. More importantly, different α subunits are widely expressed in various types of tumors and cancers, and participate in the regulation of phagocytosis, metastatic activity, cell motility and other cellular activities [ 3 ]. The dysfunction of VGSC on physiology leads to a variety of diseases and disorders [ 4 ]. In breast cancer cells, especially in highly metastatic breast cancer cells, such as MDA-MB-231 cell line, Na V 1.5 are overexpressed [ 5 ]. There are positive relations between the expression level of Na V 1.5 and the breast cancer motility, metastasis and invasiveness [ 6 ]. This indicates that Na V 1.5 may be the effective therapy targets of anticancer drugs, such as ranolazine and phenytoin [ 7 ]. Thus it has great significance and necessity in the investigations of the distributions and interactions of Na V 1.5 in normal and cancer breast cells. The mRNA and tissue sodium levels (inferred from protein-level studies, electrolyte content, clinical imaging and animal tests) have provided the evidences for the VGSC expression level in breast carcinomas and potentiation of metastasis [ 8 ]. These have also been studied by many other approaches [ 7 ]. But these investigations are from indirect means, or the spatial resolutions are limited, thus the VGSC have not been detected and localized at nanometer or single molecular spatial resolution level, the interaction forces and related binding kinetics of VGSC have not been studied at high force resolution level so far. Therefore new approaches have to be introduced in this field, the multiple and powerful nature of AFM make it the desirable toolkit in this area. AFM, an important member of Scanning Probe Microscopy, is a powerful and versatile toolkit in biotechnology [ 9 ]. AFM can image the biological samples, such as biomacromolecules (the proteins and chromatins), cell membranes, and cells at high resolution with little damages [ 10 – 12 ]. SMFS mode of AFM can measure the intra- and inter molecular forces down to picoNewton level directly, meanwhile the reaction kinetics between biomolecules can also be studied. SMFS has been applied to study the specific interaction forces in antibody-antigen and receptor-ligand successfully [ 13 , 14 ]. SMRI, the combination of AFM imaging and SMFS, has been developed to distinguish and locate the target biomolecules, such as the membrane proteins, antigens and receptors, in the heterogenic samples [ 15 ]. The recognition processes are confirmed to be highly specific, efficient and reproducible [ 16 , 17 ]. As discussed above, the multifunction and powerful features of AFM make it an appropriate toolkit in the investigations of the distributions and interactions of Na V 1.5 in breast cells. In this work, the Na V 1.5 has been specifically localized on the cellular membranes of normal and cancer breast cells at the single molecular spatial resolution level, and the interactions (including the interaction forces and binding kinetics) between the Na V 1.5 and the anti-Na V 1.5 antibody have been studied at the picoNewton force resolution level. These studies will supply innovation methods and approaches in the investigations of ion channels on cellular membranes, and will also be useful and helpful in the further studies of other membrane proteins. Materials And Methods Cell culture Hs578Bst and MDA-MB-231 cells were chosen as the representative normal and cancer breast cells, and purchased from Procell Life Science and Technology Co., Ltd (Wuhan, China). All these cells were cultured in Dulbecco’s minimum essential medium (DMEM, Biological Industries, Shanghai, China) with 10% fetal bovine serum (Biological Industries, Shanghai, China), 100 μg/mL streptomycin (Solarbio life sciences, Beijing, China) and 100 U/mL penicillin (Solarbio life sciences, Beijing, China). Cells were cultured in a humidified atmosphere with 5% CO 2 at 37 o C in incubator, and were grown as monolayer for use. Fluorescence staining and fluorescence microscopy When the cells were cultured well in the petri dish, they were washed three times by phosphate buffer saline (PBS, Corning). Then the cells were fixed with 4% paraformaldehyde for 20 min, and washed with PBS for three times. The nonspecific binding sites on cells were blocked by 2% bovine serum albumin (Solarbio life sciences, Beijing, China) for 2 h. The cells were reacted with anti-Na v 1.5 polyclonal antibody (Abcam, Shanghai, China) at 4 o C overnight. Then reacted with donkey anti-goat IgG (Alexa Fluor 488, Abcam, Shanghai, China) for 1 h in darkness. Before imaging, the cells were washed with PBS for three times to remove the unreacted dye. The fluorescence images were obtained with the laser scanning confocal microscopy LSM880 (Carl Zeiss, Shanghai, China). The Alexa Fluor 488 was excited by the laser with the wavelength of 488 nm, and the emission fluorescence (wavelength of 515 nm) was collected by the 20× or 63× objective. All the data were processed by software Zen 2.3 (Carl Zeiss, Shanghai, China). Functionalization of the AFM tips with anti - Na V 1.5 antibody The functionalization procedures were similar as described previously [ 16 ]. Briefly, anti-Na V 1.5 antibodies were reacted with N-succinimidyl 3-(acetylthio) propionate (SATP, Sigma-Aldrich, Shanghai, China). The cantilevers were cleaned in the O 3 atmosphere in ultraviolet radiation cleaner for 20 min to get rid of the organic contamination. Then the cantilevers were vapor treated with aminopropyltriethoxysilane (APTES, 99%, Sigma-Aldrich, Shanghai, China), and reacted with polyethylene glycol (PEG) crosslinkers (9.8 nm in length, MaL-PEG2000-NHS, JenKem Technology Co., Ltd., Beijing, China) in triethylamine (Sigma-Aldrich, Shanghai, China) and CHCl 3 (Richjoint Chemical, Shanghai, China). Then the cantilevers were immersed in 100 μg/mL anti-Na V 1.5 antibody with NaCNBH 3 (Sigma-Aldrich, Shanghai, China) as catalyst. In the last, 1 M ethanolamine (Sigma-Aldrich, Shanghai, China) was added to passivate the unreacted aldehyde groups. Then the modified tips were rinsed with PBS for two times and stored in PBS at 4°C until use. Atomic force microscopy All the experiments were performed with the JPK NanoWizard 4XP BioScience AFM (Bruker Corporation, Santa Barbara, California, USA). Single molecular recognition imaging was performed by the QI advanced mode, and was carried out with anti-Na V 1.5 antibody modified tips in buffer solutions at room temperature. The probes were MLCT-Bio-C (Bruker Corporation, Santa Barbara, California, USA), and the main parameters were (nominal): resonance frequency 7 kHz, spring constant 0.01 N/m. The scanning rate is 1 Hz. The recognition signals were revealed at the 75% cut-off of the background. Blocking experiments were performed by the addition of the 100 μg/mL anti-Na V 1.5 antibody into the AFM sample cell. Force spectroscopy was operated in the contact force spectroscopy mode. The deflection sensitivity of the photo-detector was determined by the slope of the force curves captured on the surfaces of cleaning silicon wafer. The actual spring constants of the cantilevers were measured by the thermal noise method in air as described previously [ 18 ]. Thousands of force curves were obtained on various positions of different cells. Blocking experiments were performed by the addition of 100 μg/mL anti-Na V 1.5 antibody into the AFM sample cell. All the AFM images and force curves were processed by the software JPK SPM Data Processing 7.0 (Bruker Corporation, Santa Barbara, California, USA). Results And Discussion The surface morphology of normal and cancer breast cells Hs578Bst and MDA-MB-231 cells are chosen as the represent normal and cancer breast cells, respectively. In order to confirm that there are Na V 1.5 on the surfaces of cells, the Na V 1.5 on the cellular membranes of Hs578Bst and MDA-MB-231 were labeled, and the fluorescent images captured on the two types of cells are shown in Fig. 1 A and 1 B, respectively. Both the Hs578Bst and MDA-MB-231 cells grow well, and there are Na V 1.5 on both types of cells. All these demonstrate that the intact and healthy Hs578Bst and MDA-MB-231 cells are obtained, and there are Na V 1.5 on the surfaces of the two types of cells. Thus the two types of cells are suitable for the next further investigations. Single molecular recognition imaging of Na V 1.5 on the surface of normal breast cells As the resolution of fluorescence microscopy is limited, we prefer single molecular recognition imaging to investigate the distributions of Na V 1.5 at single molecular resolution. In this mode, the tips were functionalized with anti-Na V 1.5 antibody via the flexible and heterobifunctional PEG crosslinker as depicted in Fig. 2 A. One anti-Na V 1.5 antibody connects only one PEG crosslinker. The nonlinear stretching characteristics of the PEG can make it to distinguish the specific events from the nonspecific ones. As the PEG is inert in chemical and physical properties, it makes the anti-Na V 1.5 antibody functionalized on the tips to reorientate rapidly and freely when the tip is approaching the surface. Meanwhile the PEG tethered anti-Na V 1.5 antibody modified on the tip can avoid to be crashed [ 9 ]. The amplitude of the cantilever is set to be less than the stretched length of the crosslinker [ 15 ]. When the Na V 1.5 sites on the cell membranes were scanned by this tip, the crosslinker will be stretched in the retraction processes of the cantilever. There will be resulting energy loss that can reduce the top peak of the oscillations. Therefore the recognition signals can be achieved and detected [ 19 ]. The recognition processes have been proved to be highly efficient, specific and reproducible [ 15 ]. The topography and corresponding recognition images captured on Hs578Bst cells are shown in Fig. 2 B and 2 C, respectively. The dark spots in Fig. 2 C represent the recognition signals that was irregularly distributed on cell membranes. The recognition signals takes a area percentage of (0.6 ± 0.2)% (N = 5) of the cell membranes. Thus for the first time we provide the quantitative information of the amount of Na V 1.5 on cell membranes by direct means. The recognition signals were superimposed onto the topographic image (shown as the blue areas in Fig. 2 B) to indicate the distributions of Na V 1.5 on the cell membranes more clearly. In order to testify that the recognition processes are specific, the blocking experiments were performed by the addition of free anti-Na V 1.5 antibody into the AFM sample cell. As the binding sites were occupied, the recognition signals disappeared as depicted in Fig. 2 D. A magnified recognition site was shown in Fig. 2 E. The amplitude reduction of the cantilever is about 3.4 nm according to the cross section analysis as shown in Fig. 2 F. However, this value in the corresponding blocked area (Fig. 2 G) is about 0.3 nm (as depicted in Fig. 2 H), which indicates that the recognition signals have been blocked efficiently. There are no recognition signals on the images acquired by the bare tips or PEG modified tips (Fig. S1 in the Supplementary Information). All these demonstrate that the recognition process is highly specific and efficient. Single molecular recognition imaging of the Na V 1.5 on the surface of cancer breast cells In order to reveal the distribution of Na V 1.5 on cancer breast cells, SMRI was performed by anti-Na V 1.5 antibody modified tips on the surface of MDA-MB-231 cells. Figure 3 A and 3 C are the topography and corresponding recognition images, respectively. Figure 3 B is the topography with the recognition signals superimposed. There are more recognition sites than that on Hs578Bst cells (as depicted in Fig. 2 C). In order to verify the specificity of the recognition process, the blocking experiment has been performed by the addition of free anti-Na V 1.5 antibody. The recognition signals disappeared as depicted in Fig. 3 D. A magnified recognition site before and after blocking is shown in Fig. 3 E and 3 G, respectively. The amplitude reduction has reduced from 2.6 nm (as depicted in Fig. 3 F) to 0.3 nm (as indicated in Fig. 3 H). The percentage of recognition areas is (7.2 ± 1.7)% (N = 5), which is about 12 times of Hs578Bst cells. This result indicates that the amount of Na V 1.5 on MDA-MB-231 cells is more than Hs578Bst cells. Force spectroscopy between Na V 1.5 on cell surfaces and the anti-Na V 1.5 antibody conjugated on the AFM tips In order to investigate the interactions between the Na V 1.5 and the anti-Na V 1.5 antibody directly, the anti-Na V 1.5 antibody was attached on the AFM tips as shown in Fig. 2 A. When the tip approaches and withdraws from the surface of cells, the interaction forces between the antibody modified on the tip and the Na V 1.5 on the cell membranes can be detected and recorded as force curves. Thousands of force curves were recorded at various positions on different cells. The typical force curve acquired on the surface of Hs578Bst cells with one unbinding event is shown in Fig. 4 A. The approaching and withdrawn processes are depicted as black and red curves, respectively. The distribution of unbinding forces is shown in Fig. 4 C. The unbinding forces range from 20–95 pN at a loading rate of 0.4 nN/s, with the maximum distribution at 52.8 ± 10.3 pN. The binding probability (the overall force curves divided by those with the specific unbinding event) is 19.6%. After blocking with free anti-Na V 1.5 antibody, the specific unbinding force event disappeared as shown in Fig. 4 B, and the binding probability has dramatically decreased to 2.8% (Fig. 4 D). There are no unbinding force events in the force curves acquired with bare tips or PEG modified tips (as depicted in Fig. 4 I). All these confirmed the unbinding forces between Na V 1.5 on the surface of Hs578Bst cells and anti-Na V 1.5 antibody on the tips are specifically and efficiently detected. The typical force curves before and after blocking recorded on the surface of MDA-MB-231 cells are shown in Fig. 4 E and 4 F, respectively. The unbinding forces are in the range of 14–90 pN at a loading rate of 0.40 nN/s. The maximum distribution is at 35.2 ± 12.5 pN as depicted in Fig. 4 G. After blocking, the specific unbinding event disappeared (as depicted in Fig. 4 F). Meanwhile the binding probability has dramatically decreased from 20.3% (Fig. 4 G) to 4.3% (Fig. 4 H). The unbinding force not only depends on the interactions between the Na V 1.5 and anti-Na V 1.5 antibody, but also depends on the loading rates of the AFM tip. The relation between the unbinding force and loading rate follows Eq. (1) according to the single barrier model. $${ F}_{u}= \frac{{k}_{B}T}{{x}_{\beta }}\text{ln}\left(\frac{r{x}_{\beta }}{{k}_{B}T{k}_{off}}\right) \left(1\right)$$ where F u is the unbinding force; k B is the Boltzmann constant; T is the thermodynamic temperature; x β is the separation energy barrier from the equilibrium position; r is the loading rate of the tip; k off is the dissociation rate constant at zero force [ 20 ]. The plots of the dependence of the unbinding forces versus loading rates are shown in Fig. 4 J. The green squares and red dots display the data recorded on Hs578Bst and MDA-MB-231 cells, respectively. From the fitting curves, it can be calculated that on Hs578Bst cells, x β = 0.20 nm, k off = 1.59 s − 1 ; on MDA-MB-231 cells, x β = 0.25 nm, k off = 2.50 s − 1 . The dissociation rate constant on Hs578Bst cells is less than MDA-MB-231 cells, which reveals that the Na V 1.5-antibody complex on Hs578Bst cells is more stable than MDA-MB-231 cells. Based on the transition state theory, the relations between activation energy ΔE and dissociation rate constant k off follow Eq. (2). $${ k}_{off}\propto \frac{-\varDelta E}{{e}^{{k}_{B}T}} \left(2\right)$$ The difference values of dissociation activation energy Δ(ΔE) of Na V 1.5-antibody complex system on Hs578Bst and MDA-MB-231 cells follows Eq. (3) [ 21 ]. $$\varDelta \left(\varDelta E\right)= -{k}_{B}T\text{l}\text{n}\left(\frac{{k}_{off}\left(Hs578Bst\right)}{{k}_{off}\left(MDA-MB-231\right)}\right) \left(3\right)$$ It can be calculated that Δ(ΔE) of Na V 1.5-antibody on Hs578Bst and MDA-MB-231 cells is 0.45 k B T . It reveals that the dissociation activation energy on Hs578Bst cells is higher than MDA-MB-231 cells, which further indicates that the Na V 1.5-antibody complex on Hs578Bst cells is more stable and harder to dissociate than MDA-MB-231 cells. Conclusion The Na V 1.5 have been detected and localized at the single molecular level on the surfaces of normal and cancer breast cells. There are more Na V 1.5 on cancer cells than normal cells (about 12 times). Thus for the first time the quantitative information on the amounts of Na V 1.5 on normal and cancer breast cells have been given. The interaction forces and binding kinetics between Na V 1.5-antibody complex system have been studied at picoNewton force resolution level. The Na V 1.5-antibody complex is stable on normal breast cells than cancer cells. The approaches and results will provide new methods in the studies of sodium channels, and will also be helpful in the further investigations of the roles of ion channels in tumor metastasis and invasion. Declarations Acknowledgements Not applicable. Author contributions XYL and LZ performed the experiments. WDZ wrote the main manuscript text. RRF, XWD, YM, YLZ, WCL and QYL prepared all figures. YHS and GWZ analyzed the data. HJZ revised the manuscript. All authors reviewed the manuscript, and read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (22004020, 61674164 and 62164001), the Natural Science Foundation of Jiangxi Province (20202ZDB01018), the open project of Key Laboratory of Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases Ministry of Education of China (XN201903), the startup funds of high-level talents of Gannan Medical University (QD202011 and QD201906) and the school level project of Gannan Medical University (YB201942). Availability of data and materials All data generated or analyzed during this study are included in this published article and supplementary information files. Consent for publication All authors agreed to submit this manuscript. Competing interests The authors have declared that no competing interest exists. Ethical Approval Not applicable. References Ruiz MD, Kraus RL: Voltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications . Journal Of Medicinal Chemistry 2015, 58 (18):7093-7118. Dong K, Du YZ, Rinkevich F, Nomura Y, Xu P, Wang LX, Silver K, Zhorov BS: Molecular biology of insect sodium channels and pyrethroid resistance . Insect Biochemistry And Molecular Biology 2014, 50 :1-17. Black Joel A, Waxman Stephen G: Noncanonical Roles of Voltage-Gated Sodium Channels . Neuron 2013, 80 (2):280-291. Bennett DL, Clark AJ, Huang JY, Waxman SG, Dib-Hajj SD: The role of voltage-gated sodium channels in pain signaling . Physiological Reviews 2019, 99 (2):1079-1151. Roger S, Gillett L, Le Guennec JY, Besson P: Voltage-gated sodium channels and cancer: is excitability their primary role? Front Pharmacol 2015, 6 . Grzywna ZJ, Borys P: On the significance of sodium ionic channels in analysis of the breast cancer metastaticity . Biosystems 2019, 177 :34-38. Angus M, Ruben P: Voltage gated sodium channels in cancer and their potential mechanisms of action . Channels 2019, 13 (1):400-409. Djamgoz MBA, Fraser SP, Brackenbury WJ: In Vivo Evidence for Voltage-Gated Sodium Channel Expression in Carcinomas and Potentiation of Metastasis . Cancers 2019, 11 (11). Kada G, Kienberger F, Hinterdorfer P: Atomic force microscopy in bionanotechnology . Nano Today 2008, 3 (1-2):12-19. Mueller DJ, Dufrene YF: Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology . Nature Nanotechnology 2008, 3 (5):261-269. Lohr D, Bash R, Wang H, Yodh J, Lindsay S: Using atomic force microscopy to study chromatin structure and nucleosome remodeling . Methods 2007, 41 (3):333-341. Cai M, Zhao W, Shang X, Jiang J, Ji H, Tang Z, Wang H: Direct Evidence of Lipid Rafts by in situ Atomic Force Microscopy . Small 2012, 8 (8):1243-1250. Hinterdorfer P, Baumgartner W, Gruber HJ, Schilcher K, Schindler H: Detection and localization of individual antibody-antigen recognition events by atomic force microscopy . Proc Natl Acad Sci U S A 1996, 93 (8):3477-3481. Florin EL, Moy VT, Gaub HE: ADHESION FORCES BETWEEN INDIVIDUAL LIGAND-RECEPTOR PAIRS . Science 1994, 264 (5157):415-417. Stroh C, Wang H, Bash R, Ashcroft B, Nelson J, Gruber H, Lohr D, Lindsay SM, Hinterdorfer P: Single-molecule recognition imaging-microscopy . Proc Natl Acad Sci U S A 2004, 101 (34):12503-12507. Jiang JG, Hao X, Cai MJ, Shan YP, Shang X, Tang ZY, Wang HD: Localization of Na+-K+ ATPases in Quasi-Native Cell Membranes . Nano Lett 2009, 9 (12):4489-4493. Wang H, Dalal Y, Henikoff S, Lindsay S: Single-epitope recognition imaging of native chromatin . Epigenetics Chromatin 2008, 1 . Butt HJ, Jaschke M: CALCULATION OF THERMAL NOISE IN ATOMIC-FORCE MICROSCOPY . Nanotechnology 1995, 6 (1):1-7. Ebner A, Kienberger F, Kada G, Stroh CM, Geretschlager M, Kamruzzahan ASM, Wildling L, Johnson WT, Ashcroft B, Nelson J et al : Localization of single avidin-biotin interactions using simultaneous topography and molecular recognition imaging . ChemPhysChem 2005, 6 (5):897-900. Evans E, Ritchie K: Dynamic strength of molecular adhesion bonds . Biophys J 1997, 72 (4):1541-1555. Yu JP, Jiang YX, Ma XY, Lin Y, Fang XH: Energy landscape of aptamer/protein complexes studied by single-molecule force spectroscopy . Chem-Asian J 2007, 2 (2):284-289. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.doc TableofContentsTOC.png Table of Contents (TOC) The cellular membranes of normal and cancer breast cells are scanned by the anti-Na V 1.5 antibody modified AFM tip, the Na V 1.5 can be localized at single molecular spatial resolution level, and the interactions between the Na V 1.5 and antibody can be detected at picoNewton force resolution level. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gaowei","middleName":"","lastName":"Zhao","suffix":""},{"id":168628148,"identity":"9a2bb28c-6bc9-4d44-a7d8-12d6982e1b6e","order_by":11,"name":"Haijian Zhong","email":"","orcid":"","institution":"Gannan Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haijian","middleName":"","lastName":"Zhong","suffix":""},{"id":168628149,"identity":"c5ba6520-5995-4b40-a0e2-f3be3414af01","order_by":12,"name":"Weidong Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDACCRDBZgPh8JCgJY10LYdJ0CI/u/nhY56y83ZrZyQwPnjbxiBvTkgL45xjxsY8524nb7uRwGw4t43BcGcDAS3MEglm0rltt5PNbiSwSfO2MSQYHCCghU0i/RtQyzmQFvbfRGnhkcgB2XLADmQLM1FaJCRyio3/nEtOMDvzsFlyzjkJww2EtMjPSN/4cEaZnb3Z8eSDH96U2cgTtAUGEhsYGBsYoNFEHLAnXukoGAWjYBSMOAAAMhU9HgBhXKoAAAAASUVORK5CYII=","orcid":"","institution":"Gannan Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Weidong","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2023-01-15 10:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2480271/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2480271/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":31815075,"identity":"058c8cc1-67b2-4221-9971-31eee8a87db1","added_by":"auto","created_at":"2023-01-19 16:16:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":219255,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence images of Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surface of Hs578Bst (A) and MDA-MB-231 (B) cells, respectively.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2480271/v1/0fae7f374695198b96ea5b5c.png"},{"id":31816228,"identity":"a7598485-041b-4d7f-9cef-40d7b6d4441b","added_by":"auto","created_at":"2023-01-19 16:24:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":729030,"visible":true,"origin":"","legend":"\u003cp\u003eSingle molecular recognition imaging of Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surface of Hs578Bst cells. (A) schematic diagram of the tip modified with anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody; (B and C) the topography and corresponding recognition images, respectively. The topography (B) with the recognition signals (blue areas) superimposed; (D) the recognition image after blocking by free anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody; (E) and (G) the magnified recognition images before and after blocking, respectively; (F) and (H) the cross section analysis along the green lines in (E) and (G), respectively.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2480271/v1/12b64efa1495f508e93095ee.png"},{"id":31816836,"identity":"93cec0bd-c958-425e-83c5-5dec7214756b","added_by":"auto","created_at":"2023-01-19 16:32:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":620620,"visible":true,"origin":"","legend":"\u003cp\u003eSingle molecular recognition imaging of Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surface of MDA-MB-231 cells. (A) and (C) the topography and corresponding recognition images, respectively; (B) the topography with the recognition signals superimposed; (D) the recognition image after blocking by free anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody; (E) and (G) the magnified recognition images before and after blocking, respectively; (F) and (H) the cross section analysis along the green lines in (E) and (G), respectively.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2480271/v1/90cceddb66bad01efe40a4a3.png"},{"id":31816227,"identity":"b63e8311-7fba-4b65-83a3-f16d03ee15ea","added_by":"auto","created_at":"2023-01-19 16:24:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125069,"visible":true,"origin":"","legend":"\u003cp\u003eForce spectroscopy between Na\u003csub\u003eV\u003c/sub\u003e1.5 and anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody on Hs578Bst and MDA-MB-231 cells. (A) and (B) the typical force curves before and after blocking acquired on the surface of Hs578Bst cells, respectively. The black and red curves represent the approaching and withdrawn processes, respectively. (A) The unbinding force event is depicted by the red arrow; (C) and (D) the histogram distributions of the unbinding force before and after blocking acquired on the surface of Hs578Bst cells, respectively; (E) and (F) the typical force curves before and after blocking acquired on the surface of MDA-MB-231 cells, respectively; (G) and (H) the histogram distributions of the unbinding force before and after blocking acquired on the surface of MDA-MB-231 cells, respectively; (I) the typical force curve acquired by bare tips or PEG modified tips; (J) the plot of the relationship of the most probable unbinding force versus the natural logarithm of the loading rate (pN/s) of the tip. The green squares and red dots are the data captured on Hs578Bst and MDA-MB-231 cells, respectively.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2480271/v1/22225efb5c6107682196398f.png"},{"id":31851567,"identity":"bdabb43e-624e-42c0-9b61-ebfc47d927d4","added_by":"auto","created_at":"2023-01-20 10:29:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2683143,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2480271/v1/3dd4cef2-f971-4e42-8371-d32569ed1267.pdf"},{"id":31815077,"identity":"ccd30b08-aa3b-4ad9-9e11-c85a2299ba6e","added_by":"auto","created_at":"2023-01-19 16:16:07","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":211968,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-2480271/v1/44ce140f35549a3f7d25f6f7.doc"},{"id":31815080,"identity":"ddfd62cd-7f6e-4068-bb6d-a65f62c43494","added_by":"auto","created_at":"2023-01-19 16:16:08","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":324422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable of Contents (TOC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cellular membranes of normal and cancer breast cells are scanned by the anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody modified AFM tip, the Na\u003csub\u003eV\u003c/sub\u003e1.5 can be localized at single molecular spatial resolution level, and the interactions between the Na\u003csub\u003eV\u003c/sub\u003e1.5 and antibody can be detected at picoNewton force resolution level.\u003c/p\u003e","description":"","filename":"TableofContentsTOC.png","url":"https://assets-eu.researchsquare.com/files/rs-2480271/v1/911d01d24d77ae109a511cc9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVGSC are transmembrane proteins that can adjust the movements of sodium ions across cellular membranes along an electrochemical gradient [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mammalian VGSC are formed by a large pseudo tetrameric pore forming α subunit (220\u0026thinsp;~\u0026thinsp;260 kDa) and one or two β subunits (33\u0026ndash;36 kDa). There are nine different α subunits (Na\u003csub\u003eV\u003c/sub\u003e1.1\u0026thinsp;~\u0026thinsp;Na\u003csub\u003eV\u003c/sub\u003e1.9) that show tissue specific expression profiles [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The opening and closing of the sodium channels are regulated by the activation and inactivation gating processes, respectively. More importantly, different α subunits are widely expressed in various types of tumors and cancers, and participate in the regulation of phagocytosis, metastatic activity, cell motility and other cellular activities [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The dysfunction of VGSC on physiology leads to a variety of diseases and disorders [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn breast cancer cells, especially in highly metastatic breast cancer cells, such as MDA-MB-231 cell line, Na\u003csub\u003eV\u003c/sub\u003e1.5 are overexpressed [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. There are positive relations between the expression level of Na\u003csub\u003eV\u003c/sub\u003e1.5 and the breast cancer motility, metastasis and invasiveness [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This indicates that Na\u003csub\u003eV\u003c/sub\u003e1.5 may be the effective therapy targets of anticancer drugs, such as ranolazine and phenytoin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus it has great significance and necessity in the investigations of the distributions and interactions of Na\u003csub\u003eV\u003c/sub\u003e1.5 in normal and cancer breast cells. The mRNA and tissue sodium levels (inferred from protein-level studies, electrolyte content, clinical imaging and animal tests) have provided the evidences for the VGSC expression level in breast carcinomas and potentiation of metastasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These have also been studied by many other approaches [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. But these investigations are from indirect means, or the spatial resolutions are limited, thus the VGSC have not been detected and localized at nanometer or single molecular spatial resolution level, the interaction forces and related binding kinetics of VGSC have not been studied at high force resolution level so far. Therefore new approaches have to be introduced in this field, the multiple and powerful nature of AFM make it the desirable toolkit in this area.\u003c/p\u003e \u003cp\u003eAFM, an important member of Scanning Probe Microscopy, is a powerful and versatile toolkit in biotechnology [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. AFM can image the biological samples, such as biomacromolecules (the proteins and chromatins), cell membranes, and cells at high resolution with little damages [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. SMFS mode of AFM can measure the intra- and inter molecular forces down to picoNewton level directly, meanwhile the reaction kinetics between biomolecules can also be studied. SMFS has been applied to study the specific interaction forces in antibody-antigen and receptor-ligand successfully [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. SMRI, the combination of AFM imaging and SMFS, has been developed to distinguish and locate the target biomolecules, such as the membrane proteins, antigens and receptors, in the heterogenic samples [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The recognition processes are confirmed to be highly specific, efficient and reproducible [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs discussed above, the multifunction and powerful features of AFM make it an appropriate toolkit in the investigations of the distributions and interactions of Na\u003csub\u003eV\u003c/sub\u003e1.5 in breast cells. In this work, the Na\u003csub\u003eV\u003c/sub\u003e1.5 has been specifically localized on the cellular membranes of normal and cancer breast cells at the single molecular spatial resolution level, and the interactions (including the interaction forces and binding kinetics) between the Na\u003csub\u003eV\u003c/sub\u003e1.5 and the anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody have been studied at the picoNewton force resolution level. These studies will supply innovation methods and approaches in the investigations of ion channels on cellular membranes, and will also be useful and helpful in the further studies of other membrane proteins.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHs578Bst and MDA-MB-231 cells\u0026nbsp;were chosen as the representative normal and cancer breast cells, and purchased from Procell Life Science and Technology Co., Ltd\u0026nbsp;(Wuhan, China).\u0026nbsp;All these cells were cultured in Dulbecco\u0026rsquo;s minimum essential medium (DMEM, Biological Industries, Shanghai, China) with 10% fetal bovine serum (Biological Industries, Shanghai, China), 100 \u0026mu;g/mL streptomycin (Solarbio life sciences, Beijing, China) and 100 U/mL penicillin (Solarbio life sciences, Beijing, China). Cells were cultured in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u003csup\u003eo\u003c/sup\u003eC in incubator, and were grown as monolayer for use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence staining and fluorescence microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the cells were cultured well in the petri dish, they were washed three times by phosphate buffer saline (PBS, Corning). Then the cells were fixed with 4% paraformaldehyde for 20 min, and washed with PBS for three times. The nonspecific binding sites on cells were blocked by 2% bovine serum albumin (Solarbio life sciences, Beijing, China) for 2 h. The cells were reacted with\u0026nbsp;anti-Na\u003csub\u003ev\u003c/sub\u003e1.5 polyclonal\u0026nbsp;antibody (Abcam, Shanghai, China) at 4\u003csup\u003eo\u003c/sup\u003eC overnight. Then reacted with\u0026nbsp;donkey\u0026nbsp;anti-goat IgG (Alexa Fluor 488, Abcam, Shanghai, China) for 1 h in darkness. Before imaging, the cells were washed with PBS for three times to remove the unreacted dye.\u003c/p\u003e\n\u003cp\u003eThe fluorescence images were obtained with the laser scanning confocal microscopy LSM880 (Carl Zeiss, Shanghai, China). The Alexa Fluor 488 was excited by the laser with the wavelength of 488 nm, and the emission fluorescence (wavelength of 515 nm) was collected by the 20\u0026times; or 63\u0026times; objective. All the data were processed by software Zen 2.3 (Carl Zeiss, Shanghai, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctionalization of the AFM tips with anti\u003c/strong\u003e-\u003cstrong\u003eNa\u003csub\u003eV\u003c/sub\u003e1.5\u003c/strong\u003e \u003cstrong\u003eantibody\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe functionalization procedures were similar as described previously\u0026nbsp;[\u003ca href=\"#_ENREF_16\" title=\"Jiang, 2009 #3\"\u003e16\u003c/a\u003e]. Briefly, anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibodies were reacted with N-succinimidyl 3-(acetylthio) propionate (SATP, Sigma-Aldrich, Shanghai, China). The cantilevers were cleaned in the O\u003csub\u003e3\u003c/sub\u003e atmosphere in ultraviolet radiation cleaner for 20 min to get rid of the organic contamination. Then the cantilevers were vapor treated with aminopropyltriethoxysilane (APTES, 99%, Sigma-Aldrich, Shanghai, China), and reacted with polyethylene glycol (PEG) crosslinkers (9.8 nm in length, MaL-PEG2000-NHS, JenKem Technology Co., Ltd., Beijing, China) in triethylamine (Sigma-Aldrich, Shanghai, China) and CHCl\u003csub\u003e3\u003c/sub\u003e (Richjoint Chemical, Shanghai, China). Then the cantilevers were immersed in 100 \u0026mu;g/mL anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody with NaCNBH\u003csub\u003e3\u003c/sub\u003e (Sigma-Aldrich, Shanghai, China) as catalyst. In the last, 1 M ethanolamine (Sigma-Aldrich, Shanghai, China) was added to passivate the unreacted aldehyde groups. Then the modified tips were rinsed with PBS for two times and stored in PBS at 4\u0026deg;C until use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAtomic force microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experiments were performed with the JPK NanoWizard 4XP BioScience AFM (Bruker Corporation, Santa Barbara, California, USA). Single molecular recognition imaging was performed by the QI advanced mode, and was carried out with anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody modified tips in buffer solutions at room temperature. The probes were MLCT-Bio-C (Bruker Corporation, Santa Barbara, California, USA), and the main parameters were (nominal): resonance frequency 7 kHz, spring constant 0.01 N/m. The scanning rate is 1 Hz. The recognition signals were revealed at the 75% cut-off of the background. Blocking experiments were performed by the addition of the 100 \u0026mu;g/mL anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody into the AFM sample cell.\u003c/p\u003e\n\u003cp\u003eForce spectroscopy was operated in the contact force spectroscopy mode. The deflection sensitivity of the photo-detector was determined by the slope of the force curves captured on the surfaces of cleaning\u0026nbsp;silicon wafer. The actual spring constants of the cantilevers were measured by the thermal noise method in air as described previously\u0026nbsp;[\u003ca href=\"#_ENREF_18\" title=\"Butt, 1995 #684\"\u003e18\u003c/a\u003e]. Thousands of force curves were obtained on various positions of different cells. Blocking experiments were performed by the addition of 100 \u0026mu;g/mL anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody into the AFM sample cell.\u003c/p\u003e\n\u003cp\u003eAll the AFM images and force curves were processed by the software JPK SPM Data Processing 7.0 (Bruker Corporation, Santa Barbara, California, USA).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch3\u003eThe surface morphology of normal and cancer breast cells\u003c/h3\u003e\n \u003cp\u003eHs578Bst and MDA-MB-231 cells are chosen as the represent normal and cancer breast cells, respectively. In order to confirm that there are Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surfaces of cells, the Na\u003csub\u003eV\u003c/sub\u003e1.5 on the cellular membranes of Hs578Bst and MDA-MB-231 were labeled, and the fluorescent images captured on the two types of cells are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, respectively. Both the Hs578Bst and MDA-MB-231 cells grow well, and there are Na\u003csub\u003eV\u003c/sub\u003e1.5 on both types of cells. All these demonstrate that the intact and healthy Hs578Bst and MDA-MB-231 cells are obtained, and there are Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surfaces of the two types of cells. Thus the two types of cells are suitable for the next further investigations.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e\u003cstrong\u003eSingle molecular recognition imaging of Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surface of normal breast cells\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAs the resolution of fluorescence microscopy is limited, we prefer single molecular recognition imaging to investigate the distributions of Na\u003csub\u003eV\u003c/sub\u003e1.5 at single molecular resolution. In this mode, the tips were functionalized with anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody \u003cem\u003evia\u003c/em\u003e the flexible and heterobifunctional PEG crosslinker as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. One anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody connects only one PEG crosslinker. The nonlinear stretching characteristics of the PEG can make it to distinguish the specific events from the nonspecific ones. As the PEG is inert in chemical and physical properties, it makes the anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody functionalized on the tips to reorientate rapidly and freely when the tip is approaching the surface. Meanwhile the PEG tethered anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody modified on the tip can avoid to be crashed [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. The amplitude of the cantilever is set to be less than the stretched length of the crosslinker [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. When the Na\u003csub\u003eV\u003c/sub\u003e1.5 sites on the cell membranes were scanned by this tip, the crosslinker will be stretched in the retraction processes of the cantilever. There will be resulting energy loss that can reduce the top peak of the oscillations. Therefore the recognition signals can be achieved and detected [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. The recognition processes have been proved to be highly efficient, specific and reproducible [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe topography and corresponding recognition images captured on Hs578Bst cells are shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, respectively. The dark spots in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC represent the recognition signals that was irregularly distributed on cell membranes. The recognition signals takes a area percentage of (0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2)% (N\u0026thinsp;=\u0026thinsp;5) of the cell membranes. Thus for the first time we provide the quantitative information of the amount of Na\u003csub\u003eV\u003c/sub\u003e1.5 on cell membranes by direct means. The recognition signals were superimposed onto the topographic image (shown as the blue areas in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) to indicate the distributions of Na\u003csub\u003eV\u003c/sub\u003e1.5 on the cell membranes more clearly. In order to testify that the recognition processes are specific, the blocking experiments were performed by the addition of free anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody into the AFM sample cell. As the binding sites were occupied, the recognition signals disappeared as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD. A magnified recognition site was shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE. The amplitude reduction of the cantilever is about 3.4 nm according to the cross section analysis as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF. However, this value in the corresponding blocked area (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG) is about 0.3 nm (as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH), which indicates that the recognition signals have been blocked efficiently. There are no recognition signals on the images acquired by the bare tips or PEG modified tips (Fig. S1 in the Supplementary Information). All these demonstrate that the recognition process is highly specific and efficient.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eSingle molecular recognition imaging of the Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surface of cancer breast cells\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eIn order to reveal the distribution of Na\u003csub\u003eV\u003c/sub\u003e1.5 on cancer breast cells, SMRI was performed by anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody modified tips on the surface of MDA-MB-231 cells. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC are the topography and corresponding recognition images, respectively. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB is the topography with the recognition signals superimposed. There are more recognition sites than that on Hs578Bst cells (as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). In order to verify the specificity of the recognition process, the blocking experiment has been performed by the addition of free anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody. The recognition signals disappeared as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD. A magnified recognition site before and after blocking is shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG, respectively. The amplitude reduction has reduced from 2.6 nm (as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF) to 0.3 nm (as indicated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH). The percentage of recognition areas is (7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7)% (N\u0026thinsp;=\u0026thinsp;5), which is about 12 times of Hs578Bst cells. This result indicates that the amount of Na\u003csub\u003eV\u003c/sub\u003e1.5 on MDA-MB-231 cells is more than Hs578Bst cells.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eForce spectroscopy between Na\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003eV\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003e1.5 on cell surfaces and the anti-Na\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003eV\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003e1.5 antibody conjugated on the AFM tips\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eIn order to investigate the interactions between the Na\u003csub\u003eV\u003c/sub\u003e1.5 and the anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody directly, the anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody was attached on the AFM tips as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. When the tip approaches and withdraws from the surface of cells, the interaction forces between the antibody modified on the tip and the Na\u003csub\u003eV\u003c/sub\u003e1.5 on the cell membranes can be detected and recorded as force curves. Thousands of force curves were recorded at various positions on different cells. The typical force curve acquired on the surface of Hs578Bst cells with one unbinding event is shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA. The approaching and withdrawn processes are depicted as black and red curves, respectively. The distribution of unbinding forces is shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC. The unbinding forces range from 20\u0026ndash;95 pN at a loading rate of 0.4 nN/s, with the maximum distribution at 52.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 pN. The binding probability (the overall force curves divided by those with the specific unbinding event) is 19.6%. After blocking with free anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody, the specific unbinding force event disappeared as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, and the binding probability has dramatically decreased to 2.8% (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). There are no unbinding force events in the force curves acquired with bare tips or PEG modified tips (as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI). All these confirmed the unbinding forces between Na\u003csub\u003eV\u003c/sub\u003e1.5 on the surface of Hs578Bst cells and anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody on the tips are specifically and efficiently detected.\u003c/p\u003e\n\u003cp\u003eThe typical force curves before and after blocking recorded on the surface of MDA-MB-231 cells are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF, respectively. The unbinding forces are in the range of 14\u0026ndash;90 pN at a loading rate of 0.40 nN/s. The maximum distribution is at 35.2\u0026thinsp;\u0026plusmn;\u0026thinsp;12.5 pN as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG. After blocking, the specific unbinding event disappeared (as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). Meanwhile the binding probability has dramatically decreased from 20.3% (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG) to 4.3% (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\n\u003cp\u003eThe unbinding force not only depends on the interactions between the Na\u003csub\u003eV\u003c/sub\u003e1.5 and anti-Na\u003csub\u003eV\u003c/sub\u003e1.5 antibody, but also depends on the loading rates of the AFM tip. The relation between the unbinding force and loading rate follows Eq.\u0026nbsp;(1) according to the single barrier model.\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equa\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$${ F}_{u}= \\frac{{k}_{B}T}{{x}_{\\beta }}\\text{ln}\\left(\\frac{r{x}_{\\beta }}{{k}_{B}T{k}_{off}}\\right) \\left(1\\right)$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003eu\u003c/em\u003e\u003c/sub\u003e is the unbinding force; \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e is the Boltzmann constant; \u003cem\u003eT\u003c/em\u003e is the thermodynamic temperature; \u003cem\u003ex\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003c/sub\u003e is the separation energy barrier from the equilibrium position; \u003cem\u003er\u003c/em\u003e is the loading rate of the tip; \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eoff\u003c/em\u003e\u003c/sub\u003e is the dissociation rate constant at zero force [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe plots of the dependence of the unbinding forces versus loading rates are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ. The green squares and red dots display the data recorded on Hs578Bst and MDA-MB-231 cells, respectively. From the fitting curves, it can be calculated that on Hs578Bst cells, \u003cem\u003ex\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003c/sub\u003e = 0.20 nm, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eoff\u003c/em\u003e\u003c/sub\u003e = 1.59 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; on MDA-MB-231 cells, \u003cem\u003ex\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003c/sub\u003e = 0.25 nm, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eoff\u003c/em\u003e\u003c/sub\u003e = 2.50 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The dissociation rate constant on Hs578Bst cells is less than MDA-MB-231 cells, which reveals that the Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody complex on Hs578Bst cells is more stable than MDA-MB-231 cells.\u003c/p\u003e\n\u003cp\u003eBased on the transition state theory, the relations between activation energy \u003cem\u003e\u0026Delta;E\u003c/em\u003e and dissociation rate constant \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eoff\u003c/em\u003e\u003c/sub\u003e follow Eq. (2).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equb\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e$${ k}_{off}\\propto \\frac{-\\varDelta E}{{e}^{{k}_{B}T}} \\left(2\\right)$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe difference values of dissociation activation energy \u003cem\u003e\u0026Delta;(\u0026Delta;E)\u003c/em\u003e of Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody complex system on Hs578Bst and MDA-MB-231 cells follows Eq.\u0026nbsp;(3) [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equc\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e$$\\varDelta \\left(\\varDelta E\\right)= -{k}_{B}T\\text{l}\\text{n}\\left(\\frac{{k}_{off}\\left(Hs578Bst\\right)}{{k}_{off}\\left(MDA-MB-231\\right)}\\right) \\left(3\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eIt can be calculated that \u003cem\u003e\u0026Delta;(\u0026Delta;E)\u003c/em\u003e of Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody on Hs578Bst and MDA-MB-231 cells is 0.45\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eT\u003c/em\u003e. It reveals that the dissociation activation energy on Hs578Bst cells is higher than MDA-MB-231 cells, which further indicates that the Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody complex on Hs578Bst cells is more stable and harder to dissociate than MDA-MB-231 cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe Na\u003csub\u003eV\u003c/sub\u003e1.5 have been detected and localized at the single molecular level on the surfaces of normal and cancer breast cells. There are more Na\u003csub\u003eV\u003c/sub\u003e1.5 on cancer cells than normal cells (about 12 times). Thus for the first time the quantitative information on the amounts of Na\u003csub\u003eV\u003c/sub\u003e1.5 on normal and cancer breast cells have been given. The interaction forces and binding kinetics between Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody complex system have been studied at picoNewton force resolution level. The Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody complex is stable on normal breast cells than cancer cells. The approaches and results will provide new methods in the studies of sodium channels, and will also be helpful in the further investigations of the roles of ion channels in tumor metastasis and invasion.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXYL and LZ performed the experiments. WDZ wrote the main manuscript text. RRF, XWD, YM, YLZ, WCL and QYL prepared all figures. YHS and GWZ analyzed the data. HJZ revised the manuscript. All authors reviewed the manuscript, and read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (22004020, 61674164 and 62164001), the Natural Science Foundation of Jiangxi Province (20202ZDB01018), the open project of Key Laboratory of Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases Ministry of Education of China (XN201903), the startup funds of high-level talents of Gannan Medical University (QD202011 and QD201906) and the school level project of Gannan Medical University (YB201942).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to submit this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interest exists.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRuiz MD, Kraus RL: \u003cstrong\u003eVoltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications\u003c/strong\u003e. \u003cem\u003eJournal Of Medicinal Chemistry \u003c/em\u003e2015, \u003cstrong\u003e58\u003c/strong\u003e(18):7093-7118.\u003c/li\u003e\n\u003cli\u003eDong K, Du YZ, Rinkevich F, Nomura Y, Xu P, Wang LX, Silver K, Zhorov BS: \u003cstrong\u003eMolecular biology of insect sodium channels and pyrethroid resistance\u003c/strong\u003e. \u003cem\u003eInsect Biochemistry And Molecular Biology \u003c/em\u003e2014, \u003cstrong\u003e50\u003c/strong\u003e:1-17.\u003c/li\u003e\n\u003cli\u003eBlack Joel A, Waxman Stephen G: \u003cstrong\u003eNoncanonical Roles of Voltage-Gated Sodium Channels\u003c/strong\u003e. \u003cem\u003eNeuron \u003c/em\u003e2013, \u003cstrong\u003e80\u003c/strong\u003e(2):280-291.\u003c/li\u003e\n\u003cli\u003eBennett DL, Clark AJ, Huang JY, Waxman SG, Dib-Hajj SD: \u003cstrong\u003eThe role of voltage-gated sodium channels in pain signaling\u003c/strong\u003e. \u003cem\u003ePhysiological Reviews \u003c/em\u003e2019, \u003cstrong\u003e99\u003c/strong\u003e(2):1079-1151.\u003c/li\u003e\n\u003cli\u003eRoger S, Gillett L, Le Guennec JY, Besson P: \u003cstrong\u003eVoltage-gated sodium channels and cancer: is excitability their primary role?\u003c/strong\u003e \u003cem\u003eFront Pharmacol \u003c/em\u003e2015, \u003cstrong\u003e6\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eGrzywna ZJ, Borys P: \u003cstrong\u003eOn the significance of sodium ionic channels in analysis of the breast cancer metastaticity\u003c/strong\u003e. \u003cem\u003eBiosystems \u003c/em\u003e2019, \u003cstrong\u003e177\u003c/strong\u003e:34-38.\u003c/li\u003e\n\u003cli\u003eAngus M, Ruben P: \u003cstrong\u003eVoltage gated sodium channels in cancer and their potential mechanisms of action\u003c/strong\u003e. \u003cem\u003eChannels \u003c/em\u003e2019, \u003cstrong\u003e13\u003c/strong\u003e(1):400-409.\u003c/li\u003e\n\u003cli\u003eDjamgoz MBA, Fraser SP, Brackenbury WJ: \u003cstrong\u003eIn Vivo Evidence for Voltage-Gated Sodium Channel Expression in Carcinomas and Potentiation of Metastasis\u003c/strong\u003e. \u003cem\u003eCancers \u003c/em\u003e2019, \u003cstrong\u003e11\u003c/strong\u003e(11).\u003c/li\u003e\n\u003cli\u003eKada G, Kienberger F, Hinterdorfer P: \u003cstrong\u003eAtomic force microscopy in bionanotechnology\u003c/strong\u003e. \u003cem\u003eNano Today \u003c/em\u003e2008, \u003cstrong\u003e3\u003c/strong\u003e(1-2):12-19.\u003c/li\u003e\n\u003cli\u003eMueller DJ, Dufrene YF: \u003cstrong\u003eAtomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology\u003c/strong\u003e. \u003cem\u003eNature Nanotechnology \u003c/em\u003e2008, \u003cstrong\u003e3\u003c/strong\u003e(5):261-269.\u003c/li\u003e\n\u003cli\u003eLohr D, Bash R, Wang H, Yodh J, Lindsay S: \u003cstrong\u003eUsing atomic force microscopy to study chromatin structure and nucleosome remodeling\u003c/strong\u003e. \u003cem\u003eMethods \u003c/em\u003e2007, \u003cstrong\u003e41\u003c/strong\u003e(3):333-341.\u003c/li\u003e\n\u003cli\u003eCai M, Zhao W, Shang X, Jiang J, Ji H, Tang Z, Wang H: \u003cstrong\u003eDirect Evidence of Lipid Rafts by in situ Atomic Force Microscopy\u003c/strong\u003e. \u003cem\u003eSmall \u003c/em\u003e2012, \u003cstrong\u003e8\u003c/strong\u003e(8):1243-1250.\u003c/li\u003e\n\u003cli\u003eHinterdorfer P, Baumgartner W, Gruber HJ, Schilcher K, Schindler H: \u003cstrong\u003eDetection and localization of individual antibody-antigen recognition events by atomic force microscopy\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e1996, \u003cstrong\u003e93\u003c/strong\u003e(8):3477-3481.\u003c/li\u003e\n\u003cli\u003eFlorin EL, Moy VT, Gaub HE: \u003cstrong\u003eADHESION FORCES BETWEEN INDIVIDUAL LIGAND-RECEPTOR PAIRS\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e1994, \u003cstrong\u003e264\u003c/strong\u003e(5157):415-417.\u003c/li\u003e\n\u003cli\u003eStroh C, Wang H, Bash R, Ashcroft B, Nelson J, Gruber H, Lohr D, Lindsay SM, Hinterdorfer P: \u003cstrong\u003eSingle-molecule recognition imaging-microscopy\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e2004, \u003cstrong\u003e101\u003c/strong\u003e(34):12503-12507.\u003c/li\u003e\n\u003cli\u003eJiang JG, Hao X, Cai MJ, Shan YP, Shang X, Tang ZY, Wang HD: \u003cstrong\u003eLocalization of Na+-K+ ATPases in Quasi-Native Cell Membranes\u003c/strong\u003e. \u003cem\u003eNano Lett \u003c/em\u003e2009, \u003cstrong\u003e9\u003c/strong\u003e(12):4489-4493.\u003c/li\u003e\n\u003cli\u003eWang H, Dalal Y, Henikoff S, Lindsay S: \u003cstrong\u003eSingle-epitope recognition imaging of native chromatin\u003c/strong\u003e. \u003cem\u003eEpigenetics Chromatin \u003c/em\u003e2008, \u003cstrong\u003e1\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eButt HJ, Jaschke M: \u003cstrong\u003eCALCULATION OF THERMAL NOISE IN ATOMIC-FORCE MICROSCOPY\u003c/strong\u003e. \u003cem\u003eNanotechnology \u003c/em\u003e1995, \u003cstrong\u003e6\u003c/strong\u003e(1):1-7.\u003c/li\u003e\n\u003cli\u003eEbner A, Kienberger F, Kada G, Stroh CM, Geretschlager M, Kamruzzahan ASM, Wildling L, Johnson WT, Ashcroft B, Nelson J\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eLocalization of single avidin-biotin interactions using simultaneous topography and molecular recognition imaging\u003c/strong\u003e. \u003cem\u003eChemPhysChem \u003c/em\u003e2005, \u003cstrong\u003e6\u003c/strong\u003e(5):897-900.\u003c/li\u003e\n\u003cli\u003eEvans E, Ritchie K: \u003cstrong\u003eDynamic strength of molecular adhesion bonds\u003c/strong\u003e. \u003cem\u003eBiophys J \u003c/em\u003e1997, \u003cstrong\u003e72\u003c/strong\u003e(4):1541-1555.\u003c/li\u003e\n\u003cli\u003eYu JP, Jiang YX, Ma XY, Lin Y, Fang XH: \u003cstrong\u003eEnergy landscape of aptamer/protein complexes studied by single-molecule force spectroscopy\u003c/strong\u003e. \u003cem\u003eChem-Asian J \u003c/em\u003e2007, \u003cstrong\u003e2\u003c/strong\u003e(2):284-289.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Voltage-gated sodium channels, breast cells, Atomic Force Microscopy, Single Molecular Recognition Imaging, Single Molecular Force microscopy","lastPublishedDoi":"10.21203/rs.3.rs-2480271/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2480271/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVoltage-gated sodium channels (VGSC) are widely expressed in various types of tumor and cancer cells, and Na\u003csub\u003eV\u003c/sub\u003e1.5 are overexpression in highly metastatic breast cancer cells. There may be positive relations between the expression levels of Na\u003csub\u003eV\u003c/sub\u003e1.5 and the breast cancer recurrence and metastasis. Herein, Na\u003csub\u003eV\u003c/sub\u003e1.5 were detected and localized on the surfaces of normal and cancer breast cells by Single Molecular Recognition Imaging mode (SMRI) of Atomic Force Microscopy (AFM). The results reveal that Na\u003csub\u003eV\u003c/sub\u003e1.5 were irregularly distributed on the surfaces of normal and cancer breast cells. The Na\u003csub\u003eV\u003c/sub\u003e1.5 take an area percentage of 0.6% and 7.2% on normal and cancer breast cells, respectively, which indicates that there are more Na\u003csub\u003eV\u003c/sub\u003e1.5 on cancer cells than normal cells. The specific interaction forces and binding kinetics between Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody complex system were investigated with Single Molecular Force Spectroscopy (SMFS) mode of AFM, indicating that the stability of the Na\u003csub\u003eV\u003c/sub\u003e1.5-antibody on normal breast cells is higher than cancer breast cells. All these results will be useful to study the interactions of other ion channel-antibody systems, and will also be useful to understand the role of sodium channels in tumor metastasis and invasion.\u003c/p\u003e","manuscriptTitle":"Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-19 16:16:02","doi":"10.21203/rs.3.rs-2480271/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8aeda424-e28c-49d5-b41a-a72a7007d71a","owner":[],"postedDate":"January 19th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-20T10:29:34+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-19 16:16:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2480271","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2480271","identity":"rs-2480271","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}