Application, Development History, and Current Status of T-Type Calcium Channel Inhibitors in Pain Treatment

The clinical management of chronic pain has long relied on traditional drugs such as opioids, anti-inflammatory drugs, and anticonvulsants, but generally faces issues of insufficient efficacy and significant side effects. There is an urgent need for novel therapeutic strategies targeting pain mechanisms. Recent studies have shown that T-type calcium channels play a key role in the development of neuropathic and inflammatory pain. Their expression and function are significantly upregulated in dorsal root ganglion and spinal dorsal horn neurons, making them a highly promising analgesic target. However, the development of direct Cav3.2 subtype channel blockers still faces challenges such as low subtype selectivity, and central nervous system and cardiovascular side effects. Currently, no ideal clinical candidate drug has emerged. This article systematically reviews the role of calcium signaling in chronic pain mechanisms, focusing on analyzing the pathological functions of T-type calcium channels (particularly the Cav3.2 subtype) and the research progress and translational challenges of their inhibitors. The aim is to provide a theoretical basis and directional guidance for developing efficient and safe pain treatment strategies. Application, Development History, and Current Status of T-Type Calcium Channel Inhibitors in Pain Treatment Fu Lu 1#, Wanbin Huang 1#, Chun Xu 1, Yanjie Zhou 1, Heyang Qin 1, and Zheman Xiao 1* 1 Department of Neurology, Renmin Hospital of Wuhan University, Wuhan 430060, China *Corresponding Author: Zheman Xiao. Department of Neurology, Renmin Hospital of Wuhan University, Wuhan 430060, China. [email protected]. # Fu Lu and Wanbin Huang have all made equal contributions to this project and are considered co-first authors.

The clinical management of chronic pain has long relied on traditional drugs such as opioids, anti-inflammatory drugs, and anticonvulsants, but generally faces issues of insufficient efficacy and significant side effects. There is an urgent need for novel therapeutic strategies targeting pain mechanisms. Recent studies have shown that T-type calcium channels play a key role in the development of neuropathic and inflammatory pain. Their expression and function are significantly upregulated in dorsal root ganglion and spinal dorsal horn neurons, making them a highly promising analgesic target. However, the development of direct Cav3.2 subtype channel blockers still faces challenges such as low subtype selectivity, and central nervous system and cardiovascular side effects. Currently, no ideal clinical candidate drug has emerged. This article systematically reviews the role of calcium signaling in chronic pain mechanisms, focusing on analyzing the pathological functions of T-type calcium channels (particularly the Cav3.2 subtype) and the research progress and translational challenges of their inhibitors. The aim is to provide a theoretical basis and directional guidance for developing efficient and safe pain treatment strategies. Key words Cav3.2; subtype selectivity; structural biology; chronic pain

Numerous studies have demonstrated the potential of T-type calcium channels in chronic pain treatment. For instance, the systematic review by Cai et al(Cai et al., 2021a) pointed out that Cav3.2 expression is significantly upregulated in primary sensory neurons in various rodent models of chronic inflammatory and neuropathic pain. Harding et al. systematically reviewed the central and peripheral contributions of T-type calcium channels to pain(Harding and Zamponi, 2022). However, these works primarily focused on the potential of T-type channels as analgesic targets and introduced related inhibitors, without providing systematic guidance for the development of T-type channel inhibitors. In recent years, the high-resolution structural elucidation of Cav3.2 by Yan Ning’s team(Huang et al., 2024), and advancements in calcium imaging technology—especially the progress of genetically encoded calcium indicators (GECIs, such as GCaMPs)(Zhang et al., 2023)—have provided unprecedented insights into the dynamic role of calcium channels in pain pathways. These developments have deepened our understanding of T-type calcium channels and offered new perspectives on the research progress and translational challenges of T-type calcium channel inhibitors. Pain is an important warning response of the body to potential or actual tissue damage. Acute pain is usually transient and self-limiting, whereas chronic pain persists or recurs for more than three months, often leading to long-term physiological dysfunction and psychological distress(Treede et al., 2019). Pain perception begins with nociceptors—a specialized class of sensory neurons responsible for detecting noxious stimuli and initiating a complex signal transduction process that ultimately forms the sensation of pain(Dubin and Patapoutian, 2010). Within this complex system, calcium channels play a central role, being crucial for the generation, transmission, and modulation of pain signals. Calcium channels are widely distributed in the nervous system and play important roles in multiple aspects of pain pathways(Park and Luo, 2010). Specifically, they regulate neurotransmitter release, neuronal excitability, and inter-synaptic signal transmission: in peripheral nociceptors, they participate in the detection and initial encoding of noxious stimuli; in dorsal root ganglia, they modulate the excitability of sensory neurons, affecting pain signal processing; at central terminals in the spinal cord, they are crucial for neurotransmitter release and the transmission of pain signals to secondary neurons. Given the multiple roles of calcium channels in pain signal transduction, they have become highly attractive targets for pain treatment. This article systematically reviews the regulatory role of calcium signaling and calcium channels in pain pathways, with a focus on summarizing the research progress and challenges in developing T-type calcium channel inhibitors. Building on this, starting from the structural and subtype characteristics of T-type calcium channels, we delve into the key factors limiting their clinical translation and identify promising research and development directions, aiming to provide new ideas and references for developing safer and more efficient chronic pain treatment strategies. Calcium Signaling and Chronic Pain Calcium ions, as key intracellular second messengers, play a central role in the occurrence and maintenance of chronic pain. Neuronal plasticity changes under chronic pain conditions, including peripheral and central sensitization, are closely related to abnormal regulation of intracellular calcium signaling(Basbaum et al., 2009; Kuner, 2010). Calcium signaling affects neuronal excitability, synaptic transmission, and gene expression through various mechanisms, thereby driving the chronic pain process. In neuropathic pain, calcium homeostasis undergoes significant changes in dorsal root ganglion (DRG) and spinal dorsal horn neurons. Nerve injury can lead to reduced voltage-dependent calcium currents, decreased resting calcium concentration, and weakened calcium transient amplitudes in DRG neurons. These changes collectively promote neuronal hyperexcitability(Fuchs et al., 2005, 2007).In diabetic neuropathy, the resting calcium concentration in sensory neurons increases, calcium extrusion capacity decreases, and the expression and function of T-type voltage-dependent calcium channels are enhanced, further exacerbating pain behavior(Fernyhough and Calcutt, 2010; Todorovic and Jevtovic-Todorovic, 2014). In chronic inflammatory pain, persistent stimulation by inflammatory mediators elevates the basal intracellular calcium concentration at sensory nerve terminals, alters voltage-dependent calcium currents, and increases neuronal excitability(Lu and Gold, 2008; Lu et al., 2010).This not only promotes neurotransmitter release from peripheral nerve terminals but also enhances transmission efficiency at central synapses, thereby maintaining the pain state(Basbaum et al., 2009). Particularly in spinal dorsal horn neurons, activation of nuclear calcium signaling has been confirmed as a key mechanism of central sensitization in inflammatory pain(Simonetti et al., 2013). Intraneuronal calcium concentration is tightly regulated, typically maintained at nanomolar levels under resting conditions(Grienberger and Konnerth, 2012). Voltage-gated calcium channels can cause a brief, significant increase in calcium concentration upon membrane depolarization. These channels are widely distributed on presynaptic and postsynaptic membranes of neurons, participating in local calcium signaling events, thereby influencing neuronal information transmission and excitability(Wheeler et al., 1994; Berridge et al., 2003).Voltage-gated calcium channels participate in rapid calcium-dependent processes such as presynaptic vesicle release and dendritic calcium spikes, and also regulate the activation of downstream signaling pathways, including long-term synaptic plasticity in the brain and spinal cord, and gene expression regulation related to neuronal growth and proliferation(Huguenard, 1996; Berridge et al., 2000; Brini et al., 2014; Leresche and Lambert, 2017). Neurons typically express multiple types of voltage-gated calcium channels, which can be classified into high-voltage-activated (HVA) channels and low-voltage-activated (LVA) channels based on their activation thresholds(Felix et al., 2013).LVA channels, also known as T-type channels, activate at more negative membrane potentials(approximately -70 to -60 mV) and include Cav3.1, Cav3.2, and Cav3.3 subtypes. Unlike HVA channels, LVA channels function as monomers containing only the α1 subunit. The pore-forming α1 subunit of voltage-gated calcium channels is encoded by 10 different genes, producing 10 unique channel subtypes. Based on differences in activation voltage, kinetic characteristics, and pharmacological properties, these channels can be categorized into different functional classes(Zamponi et al., 2015). Compared to other calcium channel families, the T-type calcium channel family has the lowest amino acid homology, and their physiological characteristics exhibit the greatest diversity. T-type channels exhibit rapidly inactivating transient currents, with an activation voltage around -60 mV, making them the most hyperpolarized activating type among all voltage-gated calcium channels in neurons, hence the name low-voltage-activated calcium channels(Fox et al., 1987; Perez-Reyes, 1998; Zamponi et al., 2015). Distribution and Function of T-type Calcium Channels The three subtypes are distributed differently in the central nervous system, peripheral neurons, and non-excitable cells, participating in distinct physiological and pathological processes (Fig. 1): Cav3.1 dominates thalamocortical rhythms and is closely related to absence seizures and sleep regulation; Cav3.2 is highly expressed in peripheral sensory neurons and the spinal dorsal horn, serving as a key mediator of pain transmission and sensitization; Cav3.3 is widely distributed in brain regions such as the hippocampus and amygdala, involved in neuronal burst firing and emotion regulation. Therefore, inhibitors lacking subtype selectivity, when intervening in specific pathological pathways, can easily cause dose-limiting side effects like drowsiness, dizziness, and cognitive impairment due to simultaneous effects on other subtypes, especially central Cav3.1 and Cav3.3. This explains why ABT-639, despite attempting peripheral restriction, still exhibited central-related adverse reactions, and why Z944, while highly effective, may potentially accompany non-targeted central effects. Cav3.1 Cav3.1 is highly enriched in specific neuronal subpopulations of the central nervous system and the sinoatrial node of the heart. In the central nervous system, for example, it is found in PV-positive GABAergic interneurons of the medial prefrontal cortex(Yabuki et al., 2025). Furthermore, it shows functional expression in hippocampal dentate gyrus neural precursor cells(Torrente et al., 2020),renal mesangial cells(Pandi et al., 2022), and various cancer tissues such as non-small cell lung cancer(Suo et al., 2018)and prostate cancer(Hu et al., 2018). Regarding neuronal function, Cav3.1 is a key molecule regulating low-threshold burst firing in PV+ neurons. Its absence significantly reduces burst firing frequency, affecting the excitation-inhibition balance in the prefrontal cortex(Yabuki et al., 2025). Cav3.1 dysfunction is associated with various diseases, including depression, age-related hearing loss(Pan et al., 2021), and the proliferation and invasion of various tumors(Hu et al., 2018; Suo et al., 2018; Li et al., 2021). In the cardiac sinoatrial node, Cav3.1 is the main T-type calcium channel involved in regulating cardiac automaticity(Torrente et al., 2020). This reasonably explains why non-selective inhibition of T-type calcium channels easily leads to unacceptable side effects like bradycardia, drowsiness, and cognitive impairment(Suo et al., 2018; Torrente et al., 2020). Therefore, targeting T-type calcium channels as a systemic analgesic strategy faces severe challenges. Developing highly selective T-type calcium channel modulators must focus on solving issues of brain region-targeted delivery or state-dependent modulation, with the aim of separating their potential central analgesic effects from broad nervous system and cardiovascular side effects. Cav3.2 Cav3.2 is particularly prominent in pain pathways of the peripheral and central nervous systems. It is highly expressed in small-diameter nociceptive neurons of the dorsal root ganglia(Cai et al., 2024)and trigeminal ganglia(Zhang et al., 2021), closely associated with pain. In the central nervous system, it is also distributed in the thalamus, hippocampus(Mustafá et al., 2023), and some GABAergic neurons in the prefrontal cortex(Liang et al., 2025). Additionally, it is expressed in platelets(Tamang et al., 2022), hypothalamic arcuate nucleus neurons(Feng et al., 2021), and various inflammatory and tumor environments(Hiramoto et al., 2020). In pain signal transduction, Cav3.2 is a core mediator of chronic pain(including neuropathic, inflammatory, and visceral pain), as detailed in the review by Cai et al(Cai et al., 2021b). Its upregulation or enhanced activity significantly increases the excitability of sensory neurons, promoting pain sensitization(Zhang et al., 2021; Cai et al., 2024). There is also evidence that Cav3.2 abnormality is involved in regulating autism-like behaviors(Liang et al., 2025) and regulates feeding and energy metabolism in hypothalamic neurons, affecting obesity development(Feng et al., 2021). Cav3.3 Cav3.3 expression shows high brain region specificity, with its most notable feature being high expression in GABAergic neurons of the thalamic reticular nucleus (TRN)(El Ghaleb et al., 2025). It is also distributed in the hippocampal CA1 region(Mustafá et al., 2023), cerebellar Purkinje cells(Eickhoff et al., 2022), retinal ganglion cells(Wang et al., 2023), and splenic T cells(Ye and Liu, 2025). Cav3.3 primarily influences sleep rhythms and thalamic function. It is a key ion channel for generating and maintaining spindle wave rhythms during non-rapid eye movement sleep(El Ghaleb et al., 2025) and is crucial for the rhythmic pacemaker activity of thalamic neurons. Its function is finely regulated by the constitutive activity of dopamine D5 receptors(Mustafá et al., 2023), thereby affecting firing patterns of neurons in brain regions like the hippocampus, and is associated with epileptogenesis. Furthermore, studies show that Cav3.3 regulates exercise-induced calcium signaling and immune function in T cells(Ye and Liu, 2025); in a glaucoma model, its upregulation mediates calcium overload and apoptosis in retinal ganglion cells(Wang et al., 2023). This heterogeneity in distribution and function means that ideal analgesic drugs should act highly selectively on Cav3.2 while minimizing inhibition of Cav3.1 and Cav3.3, to separate analgesic effects from cardiovascular, central rhythm, and cognitive side effects. Evidence for Cav3.2 Upregulation in Chronic Pain During the development of chronic pain, the upregulation of Cav3.2 expression and function has become an important molecular basis for pain signal amplification and maintenance. Multiple studies have confirmed this upregulation phenomenon in different types of chronic pain models and revealed its mechanism of involvement in pain sensitization (Fig .2).In chronic pain states, the expression and function of Cav3.2 are often upregulated. This upregulation is primarily regulated through post-translational modifications, including glycosylation, phosphorylation, and ubiquitination. These modifications significantly enhance the membrane expression or electrophysiological activity of Cav3.2 in pain models, thereby promoting neuronal hyperexcitability and pain hypersensitivity. In various neuropathic pain models, Cav3.2 shows upregulated expression and function at both DRG and spinal levels. After chronic constriction injury of the sciatic nerve, the amplitude of Cav3.2 current in DRG neurons is significantly enhanced(Jagodic et al., 2008). In diabetic peripheral neuropathy models, Cav3.2 current in DRG neurons is enhanced, and in vivo silencing of Cav3.2 alleviates tactile allodynia(Jagodic et al., 2007; Messinger et al., 2009). In chronic compression of DRG models, Cav3.2 mRNA expression increases, and intrathecal administration of its antisense oligonucleotide alleviates allodynia(Wen et al., 2006, 2010). In L5/6 spinal nerve ligation models, Cav3.2 protein expression increases in the spinal cord and DRG, and T-type calcium channel antagonists alleviate pain(Chen et al., 2015). In spared nerve injury models, upregulated Cav3.2 protein expression is observed, accompanied by enhanced afterdepolarization of action potentials(Kang et al., 2018). After partial sciatic nerve ligation, the mRNA, protein, and current density of Cav3.2 significantly increase in superficial spinal dorsal horn neurons(Feng et al., 2019). In trigeminal neuralgia models, Cav3.2 activity also increases in trigeminal ganglion neurons(Gomez and Khanna, 2022). In chemotherapy-induced and metabolic disorders such as paclitaxel-induced peripheral neuropathy, DRG neuron excitability increases, and Cav3.2 expression transiently rises(Li et al., 2017).In inflammatory conditions such as complete Freund’s adjuvant-induced chronic inflammation models, Cav3.2 expression increases at both mRNA and protein levels(Lin et al., 2016). In knee osteoarthritis pain models, Cav3.2 protein expression increases in sensory neurons and spinal dorsal horn, and T-type calcium current amplitude more than doubles(Shin et al., 2020).In patients with irritable bowel syndrome and animal models, Cav3.2 expression and membrane trafficking increase in colonic mucosal nerve fibers or sensory neurons, and T-type calcium current amplitude increases(Scanzi et al., 2016). Glycosylation of the Cav3.2 channel occurs mainly through the addition of sugar chains to asparagine residues, facilitating proper protein folding, trafficking to the cell membrane, and enhancing its stability. In pain models such as diabetic peripheral neuropathy, the glycosylation level of Cav3.2 is elevated, leading to increased current and pain hypersensitivity. Blocking glycosylation can alleviate pain behaviors without affecting normal pain thresholds(Orestes et al., 2013; Weiss et al., 2013).Furthermore, cyclin-dependent kinase 5 (CDK5) can phosphorylate Cav3.2, increasing its current density. In neuropathic pain models, CDK5 expression is upregulated, and its inhibitors reduce T-type current in dorsal root ganglion neurons and partially reverse pain behaviors(Gomez et al., 2020). This suggests that CDK5-mediated phosphorylation is an important mechanism for Cav3.2 functional upregulation.Moreover, Cav3.2 can be tagged by ubiquitination for degradation, while the deubiquitinase USP5 removes ubiquitin chains, stabilizing Cav3.2 and increasing its membrane expression. In chronic inflammatory and neuropathic pain models, USP5 expression is elevated in the dorsal root ganglia and spinal cord, enhancing Cav3.2-mediated presynaptic calcium influx and neurotransmitter release. Inhibiting USP5 activity or disrupting its interaction with Cav3.2 significantly alleviates pain(García-Caballero et al., 2014; Gadotti et al., 2015).Together, these mechanisms coordinately regulate Cav3.2 upregulation in chronic pain, providing a molecular basis for its potential as a therapeutic target. In summary, under various pathological conditions such as neuropathic, inflammatory, metabolic, and visceral chronic pain, Cav3.2 channel expression and function are commonly upregulated in sensory pathways. By enhancing neuronal excitability, it participates in pain sensitization processes, thus serving as a key molecule in the occurrence and maintenance of chronic pain. T-type Calcium Channel Inhibitors Classical Inhibitors: Z944, TTA-P2, ABT-639 In the development of T-type calcium channel inhibitors, classical drugs like TTA-P2, ABT-639, and Z944 have demonstrated different pharmacological properties and limitations. TTA-P2 is a class of synthetic small-molecule T-type calcium channel inhibitors designed to develop high-selectivity, centrally penetrant T-type channel(Choe et al., 2011). In various pain models, TTA-P2 exhibits significant anti-allodynic and anti-hyperalgesic effects, particularly in peripheral nerve injury and inflammation models. Its application in pain research stems from the key role of T-type channels in pain sensitization. Regarding analgesic efficacy, in spinal cord injury-related neuropathic pain models, it demonstrates analgesic effects superior to gabapentin(Liu et al., 2023). Despite its clear analgesic effects in animal models, TTA-P2 itself has not been developed into a relevant drug for clinical treatment. It has primarily remained a highly selective research tool compound and has not entered clinical development stages. Currently, TTA-P2 is mainly used in preclinical research as a tool to investigate the role of T-type channels in physiological and pathological processes like pain, epilepsy, and sleep. Its structural scaffold also provides an important reference for the subsequent design of more selective inhibitors. ABT-639 is a peripherally restricted Cav3.2 selective inhibitor designed to avoid central nervous system side effects related to brain function(Zhang et al., 2015). Its design concept stems from the high expression of T-type channels in peripheral nociceptive neurons, especially the central role of Cav3.2 in pain signal conduction(Jarvis et al., 2014). ABT-639 performed excellently in the preclinical stage and entered Phase II clinical trials for treating diabetic peripheral neuropathic pain(Ziegler et al., 2015). However, it caused some adverse reactions such as dizziness and nausea, ultimately failing to meet the primary endpoint. Concurrently, another study in tsA-201 cells found that 30 μM ABT-639 inhibited less than 15% of Cav3.2 current, whereas 1 μM Z944 inhibited about 50% of the current(Antunes et al., 2024). This directly explains the reason for its clinical failure. The clinical development of ABT-639 has been terminated, making it a classic case of T-type channel inhibitor transition from preclinical success to clinical failure(Ziegler et al., 2015; Zamponi, 2016). Z944, as a blood-brain barrier permeable, highly selective T-type calcium channel antagonist, simultaneously addresses the issues of low target selectivity and insufficient analgesic efficacy, providing a new targeted approach for pain treatment. Z944 was initially developed as a highly selective, centrally penetrant T-type calcium channel antagonist for treating absence seizures(Casillas-Espinosa et al., 2015). In recent years, given the important role of T-type calcium channels in pain pathways, the research focus on Z944 has expanded to chronic pain, including neuropathic and inflammatory pain(Tringham et al., 2012). In Harding et al.’s experiment, in a CFA-induced inflammatory model, intraperitoneal administration showed that the IC50 of Z944 for anti-allodynia in this model was 1.76 ± 0.14 mg/kg, indicating potent dose-dependent analgesic effects(Harding et al., 2021). Furthermore, in non-painful normal rats, equivalent doses of Z944 did not alter baseline mechanical pain thresholds, indicating that its analgesic effect is selective for pathological states rather than simple sedation or motor inhibition. However, given that this study involved systemic administration, central inhibition could not be ruled out. Intrathecal injection of Z944 in chronic pain rodent models followed by assessment of pain thresholds and behavior could confirm that Z944 exerts its analgesic effect by acting on spinal T-type channels. Research on Z944 is mainly in the late preclinical stage. Since Phase I trials around 2014(Lee, 2014), publicly available data on Z944 entering Phase II or III pain clinical trials are very limited. Its clinical development path does not seem to have progressed as rapidly as initially expected. In an epilepsy study, Z944 exhibited dose-dependent sedative side effects at 100 mg/kg(Casillas-Espinosa et al., 2015), likely related to systemic inhibition of Cav3.1 due to its central penetrance. To elucidate its mechanism, researchers successfully resolved the high-resolution structure of human Cav3.1 channel using cryo-electron microscopy—the apo state (3.3 Å) and the Z944-bound state (3.1 Å)(Zhao et al., 2019). Structural analysis visually revealed that Z944 binds to the central cavity of the channel in an arch-like conformation, with one end of the molecule inserting into the lateral fenestration of the pore and the other end directly blocking above the cytoplasmic gate, confirming the direct pore-blocking effect of this compound on the Cav3.1 channel at the atomic level. This structural discovery not only provides a molecular basis for understanding the pharmacological action of Z944 but may also offer a structural explanation for the observed sedative side effects in its clinical application. Next-generation Inhibitors Based on Structural Biology: ACT-709478 With the development of structural biology, understanding of the mechanism of action of T-type calcium channel inhibitors has deepened. Yan Ning’s team resolved the high-resolution cryo-EM structures of human Cav3.2 protein bound to four selective antagonists: ACT-709478, TTA-A2, TTA-P2, and ML218(Huang et al., 2024). ACT-709478, also known as NBI-827104, is an orally effective selective T-type voltage-gated calcium channel antagonist currently in clinical research stages for neurological diseases. ACT-709478 was initially reported as a potent, selective T-type calcium channel blocker developed as a candidate drug for treating generalized epilepsy(Bezençon et al., 2017). Surprisingly, although ACT-709478 lacks subtype selectivity, it showed good safety in first-in-human assessment, with characteristics such as long half-life, dose-non-proportional absorption, minimal food effect, and significant correlation between saliva and plasma concentrations(Richard et al., 2019). Central nervous system-related abnormalities were only mild, with no cardiovascular dysfunction. Similar results were obtained in another multiple-dose escalation study(Richard et al., 2020), providing an important foundation for its subsequent development as an epilepsy drug. Similar to Z944, its analgesic efficacy remains to be studied. By reviewing classical T-type calcium channel inhibitors like Z944, TTA-P2, and ABT-639, a clear research and development evolution trajectory can be observed: from early pursuits of high selectivity and central penetrance like Z944 and TTA-P2, to attempting to avoid central side effects through peripheral restriction design like ABT-639, researchers have continuously explored safer and more effective pain treatment strategies. Although these molecules have shown promising analgesic potential in preclinical models and gradually enhanced our understanding of the role of T-type channels in pain, they collectively highlight a core scientific bottleneck yet to be broken: the lack of high selectivity for inhibiting different T-type calcium channel subtypes (Cav3.1, Cav3.2, Cav3.3). This limitation is not accidental; its root lies in the high structural and functional similarity among T-type calcium channel subtypes. Structure of T-type Calcium Channels From a structural biology perspective, the fundamental reason for the difficulty in achieving subtype selectivity lies in the high conservation of the transmembrane core regions among the three subtypes, especially the key amino acid sequences of the pore region and voltage-sensing domain, which share extremely high homology. This makes it difficult for small-molecule inhibitors to find subtype-specific binding pockets with sufficient distinction. The T-type calcium channel α1 subunit is encoded by three different genes, CACNA1G, CACNA1H, and CACNA1I, corresponding to the Cav3.1, Cav3.2, and Cav3.3 channel subtypes, respectively. These three T-type channels are highly similar but exhibit subtle differences in activation voltage, sensitivity to inhibition by divalent cations such as nickel, cadmium, zinc, and specific T-type channel antagonists(Huguenard, 1996; Zamponi et al., 1996, 1996; Lee et al., 1999). In various neurons, inhibiting T-type channels or reducing their expression through gene knockout or knockdown techniques decreases neuronal excitability(Wheeler et al., 1994; Dubreuil et al., 2004; Cain and Snutch, 2010; Tscherter et al., 2011; Joksimovic et al., 2017; Candelas et al., 2019). In recent years, Yan Ning’s team successfully resolved the high-resolution three-dimensional structure of human Cav3.2 channel using cryo-electron microscopy. High-resolution cryo-EM studies reveal that T-type calcium channel antagonists typically bind to the central cavity of the pore, directly blocking ion flow (Fig. 3). This milestone work revealed the atomic-level structural details of this subtype for the first time, including its unique extracellular loop regions and potential drug-binding sites, providing a key framework for understanding Cav3.2 function and regulation. However, breakthroughs in structural biology have not directly translated into progress in developing subtype-selective inhibitors. The main reasons include: First, structural differences between Cav3.1 and Cav3.2 are mainly concentrated in relatively flexible extracellular loop regions, whereas traditional small-molecule drugs typically target more conserved transmembrane regions. Second, structure-based virtual screening and rational design are still limited by subtle differences in ligand-binding pockets between subtypes, which are more obscure during channel dynamics and allosteric modulation processes. Third, translating structural information into actual drugs still needs to overcome multiple pharmaceutical challenges such as compound synthesis, membrane permeability, and metabolic stability. Additionally, drug binding may depend on the surrounding lipid environment, adding complexity to in vivo efficacy prediction. Therefore, a deep understanding of the fine structure of T-type channels is a prerequisite for developing the next generation of highly selective inhibitors and is key to overcoming their clinical translation bottleneck. Thus, current research and development of T-type calcium channel inhibitors are in a period of ”clear structure, selectivity still difficult.” Yan Ning team’s research undoubtedly lays a solid foundation for precise drug design targeting Cav3.2. However, to truly achieve subtype-selective inhibition, it is still necessary to integrate dynamic conformational analysis, exploration of allosteric sites, and strategies for macromolecules or allosteric modulators targeting subtype-specific extracellular domains. In the future, interdisciplinary collaboration integrating structural biology, computational chemistry, and neuropharmacology is expected to break through this long-standing selectivity bottleneck and develop truly safe and effective subtype-specific T-type channel therapies. Novel GCaMP Calcium Indicators Assisting T-type Calcium Channel Research GCaMP calcium indicators, as the current mainstream genetically encoded calcium imaging tools, are of great significance for in-depth research on the role of T-type calcium channels in pain pathways. By coupling calcium ion binding with fluorescent signal output, GCaMP enables real-time, cell-type-specific observation of neuronal calcium activity in live animals, providing a key means to analyze the dynamic function of T-type calcium channels in pain transmission and modulation(Nakai et al., 2001; Chen et al., 2013) . In recent years, the GCaMP series has been continuously optimized, with the latest generation, GCaMP8, showing significant improvements in sensitivity, response speed, and photobleaching resistance (Zhang et al., 2023). This provides a more powerful tool for high-fidelity, long-term monitoring of subtle calcium signals mediated by T-type channels in complex pain models. Due to their low-voltage activation characteristics, T-type calcium channels play important roles in neuronal excitability, burst firing, and pain sensitization, especially being upregulated in various chronic pain models such as neuropathic pain, inflammatory pain, and diabetic neuropathy(Bourinet et al., 2005; Jagodic et al., 2007). Utilizing GCaMP imaging technology, researchers can directly capture calcium transients mediated by T-type channel activity in live systems, thereby revealing their spatiotemporal activation patterns under specific pain states. For example, a study using GCaMP6m found through fiber photometry recording that remifentanil-induced upregulation of T-type calcium channel function enhances thalamocortical pathway activity, promoting the development of secondary hyperalgesia(Jin et al., 2022). Furthermore, the high spatiotemporal resolution of GCaMP helps elucidate the regulatory mechanisms of T-type channels, such as the effects of post-translational modifications like glycosylation and phosphorylation on their function and membrane localization(Orestes et al., 2013; Gomez et al., 2020). Therefore, GCaMP not only promotes empirical research on the role of T-type calcium channels in pain but also provides an important methodological foundation for developing new analgesic strategies targeting this channel. A Novel Natural Product-derived Inhibitor: Buxus Alkaloids? Novel Cav3.2 inhibitors like Buxusemine L (BXSL) represent a new direction for natural products in targeted therapy(Gong et al., 2024). Gong Y et al. found that the inhibitory activity of BXSL against Cav3.2 is comparable to Z944, while its selectivity for Cav3.1 is increased by about 30-fold. Through point mutation experiments and molecular simulation, the study further clarified that BXSL binding to Cav3.2 depends on hydrogen bonding with the S1543 residue in domain III, whereas the corresponding position in Cav3.1 is alanine (Fig .4). This provides a key structural basis for designing highly selective Cav3.2 inhibitors. In terms of analgesic effects, 5 mg/kg of BXSL significantly reduced action potential frequency in mouse dorsal root ganglion neurons, attenuating pain signal conduction. Its analgesic effect was equivalent to 10 mg/kg of Z944 or mibefradil. Compared to non-selective inhibitors, BXSL showed no significant effect on nociceptive ion channels like TRPV1 and Nav1.7, indicating low off-target risk. More importantly, it can preserve Cav3.1 function, potentially reducing cardiovascular or nervous system side effects caused by broad inhibition of T-type calcium channels, thus representing an important advancement in this field. Researchers have systematically isolated extracts from the leaves and branches of Buxus sempervirens, revealing the cardioprotective potential of buxusemine series compounds(Xiang et al., 2021). However, because Cav3.3 also has a conserved serine residue at the corresponding position (S1348), BXSL does not show high selectivity between Cav3.2 and Cav3.3. Although BXSL exhibits higher subtype selectivity compared to Z944, its inhibitory activity against Cav3.3 is similar to that against Cav3.2, and subtype selectivity still needs further improvement; otherwise, potential safety issues may arise. Nonetheless, the work by Gong Y et al. successfully identified a class of structurally novel analgesic compounds with relatively high selectivity for Cav3.2 and revealed their unique binding mode, laying an important theoretical foundation for the development of next-generation structure-based Cav3.2-targeted drugs. Future drugs optimized based on this are expected to possess better safety and efficacy. Additionally, other natural product-derived scaffolds have shown promise as selective Cav3.2 inhibitors. Betulinic acid and the synthetic compound 5bk have been identified to selectively inhibit Cav3.2 channels over other voltage-gated calcium channels. They demonstrated efficacy in reversing mechanical allodynia in diverse preclinical models, including paclitaxel-induced, HIV-associated, and spinal nerve ligation-induced neuropathic pain(Bellampalli et al., 2019; Cai et al., 2020). These findings underscore the potential of pharmacophore discovery from natural sources in advancing Cav3.2-targeted analgesic drug development.This has been described in detail in the review by Cai et al(Cai et al., 2021b). Summary and Outlook In summary, within the pain signal transmission and modulation network composed of primary afferent neurons in dorsal root ganglia, superficial neurons in the spinal dorsal horn, and brain regions such as the anterior cingulate cortex/prefrontal cortex, Cav3.2 T-type calcium channels have been confirmed as a core molecular mechanism mediating various chronic and refractory pain pathological processes. This channel plays a key role in peripheral sensitization, central sensitization, and even the emotional dimension of pain by regulating neuronal excitability thresholds, burst firing patterns, and synaptic plasticity. Therefore, specific inhibition targeting Cav3.2 is considered a highly promising analgesic strategy, and its development has entered a critical stage of translational exploration from preclinical validation. A breakthrough could fundamentally change the current pain management dilemma dominated by symptomatic treatment. Current first-line clinical analgesics, such as opioids and non-steroidal anti-inflammatory drugs,generally faces issues of single mechanism, limited efficacy, high risk of tolerance and addiction, and prominent effects with long-term use. Although first-generation T-type calcium channel blockers showed potential effects on the cardiac conduction system and thalamocortical rhythms in clinical trials, these side effects stemmed from their cross-inhibition of subtypes like Cav3.1 and could theoretically be avoided by improving subtype selectivity. Reviewing its development path, from broad-spectrum T-type blockers like mibefradil and TTA-P2, which were withdrawn due to cardiovascular side effects, to the relatively T-type channel selective Z944 that entered Phase II clinical trials, to recent lead compounds designed for the Cav3.2 subtype, research in this field has achieved a leap from non-selectivity to subtype selectivity, with simultaneous improvement in analgesic efficacy and safety margins (Table .1). Particularly crucial, the high-resolution structures of Cav3.1/3.3 resolved by Yan Ning’s team provide an atomic-level blueprint for understanding the gating mechanism, ion selectivity, and drug-binding sites of T-type calcium channels. This structural information is guiding computationally rational drug design aimed at developing next-generation targeted Cav3.2 therapeutic drugs that combine high subtype selectivity, potent analgesic activity, and good systemic safety. In this context, the emergence of novel Cav3.2 inhibitors like Buxusemine L (BXSL) is leading the field towards a new stage of more precise subtype-specific modulation(Table .1). As a class of highly selective Cav3.2-targeted candidate molecules, BXSL has demonstrated significant efficacy in inhibiting chronic pain in preclinical models while minimizing interference with subtypes like Cav3.1, thereby theoretically avoiding cardiovascular and central nervous system side effects associated with early T-type channel blockers. It not only represents an important advancement in lead compound optimization but also validates the feasibility of rational drug design based on structural biology, providing a key proof-of-concept for developing truly ”clean” Cav3.2 inhibitors. Furthermore, the elaborate regulatory network controlling Cav3.2 expression and function (including transcription, phosphorylation, ubiquitination, and glycosylation) unveils a new frontier for therapeutic intervention. Future research should parallelly explore “indirect” strategies targeting these regulatory nodes (e.g., Egr-1, USP5, Cdk5) alongside the optimization of direct pore blockers. This dual-path approach may yield combination therapies or context-dependent therapeutics that are effective across different etiologies of chronic pain. The development of biomarkers related to specific Cav3.2 post-translational modifications (e.g., glycosylation status in diabetes) could further enable personalized analgesic strategies. Future breakthroughs will rely on deep integration of multiple disciplines: structural biology for resolving precise drug-channel binding modes; computational chemistry and artificial intelligence to assist in designing highly selective molecules; translational medical research focusing on building disease models that better predict human responses and systematically investigating the influence of factors such as gender and genetic background. Ultimately, successful Cav3.2-targeted therapy is likely not a single molecule but a ”precision toolbox” containing specific inhibitors or modulators for different pain types and patient subgroups, achieving true individualized analgesia. The continuous development and optimization of highly selective inhibitors like BXSL are the core driving force for building this toolbox and advancing it towards clinical application. Acknowledgments We thank Figdraw for providing the materials for the figures. The National Natural Science Foundation of China (82471577), Major and stubborn disease project of national administration of traditional Chinese medicine Interdisciplinary (ZDYN-2024-A-094). Innovative Talents Foundation of Renmin Hospital at Wuhan University (JCRCYG-2022-006), Research on Degree and Graduate Education Teaching Reform at Wuhan University, Teaching Research Project of Wuhan University Medical Science Center (2024YB08) have funded this research.Undergraduate Training Programs forInnovation of Wuhan University(202510486153). Declarations Competing interests: The authors declare no conflicts of interest regarding the publication of this paper. Ethics approval and consent to participate: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials: Not applicable. Word:5437 Antunes, F.T.T., Huang, S., Chen, L., and Zamponi, G.W. (2024). Effect of ABT-639 on Cav3.2 channel activity and its analgesic actions in mouse models of inflammatory and neuropathic pain. Eur J Pharmacol 967 : 176416.Basbaum, A.I., Bautista, D.M., Scherrer, G., and Julius, D. (2009). 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(2015). A randomized double-blind, placebo-, and active-controlled study of T-type calcium channel blocker ABT-639 in patients with diabetic peripheral neuropathic pain. Pain 156 : 2013–2020. Figure 1 . Distribution and Physiological Functions of T-type Calcium Channels.Distribution and functional associations of Cav3.1, Cav3.2 and Cav3.3 in distinct brain regions (prefrontal cortex, thalamus, hippocampus, etc.) and peripheral tissues (sinoatrial node, DRG).Fig 1 is drawn by Figdraw. Figure 2. Mechanisms of Cav3.2 upregulation in chronic pain. Increased Cav3.2 expression in the dorsal root ganglion enhances neuronal excitability, thereby contributing to pain sensitization.Fig 2 is drawn by Figdraw. Figure 3. Binding modes of selective antagonists to T-type calcium channel Cav3.2.The structure illustrates two distinct binding poses of four representative antagonists (ACT-709478, TTA-A2, TTA-P2, ML218) within the central cavity of Cav3.2. ACT-709478 and TTA-A2 bind to the IV–I fenestration, whereas TTA-P2 and ML218 occupy the II–III fenestration. An α→π helical transition in the S6Ⅱ segment is observed upon antagonist binding, suggesting a conformational change associated with state-dependent inhibition.(Image adapted from: Huang et al., 2024, Cell research). Figure 4. Simulated binding mode of Buxusemine L (BXSL) to the Cav3.2 channel.This figure illustrates the molecular docking and dynamics simulation of the 9(10/19)abeco-artane-type Buxus alkaloid BXSL complexed with human Cav3.2. (A–B) BXSL (dark red sticks) occupies the central cavity within the DII–DIII fenestration of Cav3.2. The aminomethyl group at C3 forms a critical hydrogen bond (black dashed line) with Ser1543 in the S6Ⅲ segment, which underlies its interaction diagram further details the hydrophobic contacts and hydrogen‑bond network between BXSL and channel residues. (D) A surface representation viewed from the extracellular side shows the spatial occupation of BXSL within the Cav3.2 pore. These results reveal a novel binding mode of a Cav3.2‑selective inhibitor and provide a structural template for structure‑based drug design targeting Cav3.2.(Adapted from Gong et al.,2024.Angew. Chem. Int. Ed). Table 1 . Comparative Analysis of T-type Calcium Channel (Cav3.2)-Targeted Analgesic Drug. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Metrics & Citations Metrics Article Usage 482views 123downloads Citations Download citation Fu Lu, Wanbin Huang, Chun Xu, et al. Application, Development History, and Current Status of T-Type Calcium Channel Inhibitors in Pain Treatment. Authorea. 19 December 2025. 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