Α2δ Subunits Research Articles

The Evidence for Effective Inhibition of INa Produced by Mirogabalin ((1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0] hept-3-ene-6-acetic acid), a Known Blocker of CaV Channels.

Mirogabalin (MGB, Tarlige®), an inhibitor of the α2δ-1 subunit of voltage-gated Ca2+ (CaV) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH3) cells, we found that MGB was effective at suppressing the peak (transient, INa(T)) and sustained (late, INa(L)) components of the voltage-gated Na+ current (INa) in a concentration-dependent manner, with an effective IC50 of 19.5 and 7.3 μM, respectively, while the KD value calculated on the basis of minimum reaction scheme was 8.2 μM. The recovery of INa(T) inactivation slowed in the presence of MGB, although the overall current–voltage relation of INa(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (INa(W)) or resurgent INa (INa(R)) evoked by the respective ascending or descending ramp pulse (Vramp) was reduced during cell exposure to MGB. MGB-induced attenuation in INa(W) or INa(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate INa. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent INa in response to the isosceles triangular Vramp. The cumulative inhibition of INa(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of CaV channels, the NaV channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo.

Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1.

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as a voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α1S subunit and the auxiliary α2δ-1 and γ1 subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing α2δ-1, β3, and STAC3. When the adult (CaV1.1a) and embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further coexpressed the γ1 subunit was the current density of CaV1.1a, but not that of CaV1.1e, reduced by >50%. In addition, γ1 caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of γ1, unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the γ1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the γ1-dependent reduction of current density, suggesting that structural rearrangements in CaV1.1a induced by inclusion of exon 29 may allosterically empower the γ1 subunit to exert its inhibitory action on CaV1.1 calcium currents.

ADAM17 Mediates Proteolytic Maturation of Voltage-Gated Calcium Channel Auxiliary α2δ Subunits, and Enables Calcium Current Enhancement.

The auxiliary α2δ subunits of voltage-gated calcium (CaV) channels are key to augmenting expression and function of CaV1 and CaV2 channels, and are also important drug targets in several therapeutic areas, including neuropathic pain. The α2δ proteins are translated as preproteins encoding both α2 and δ, and post-translationally proteolyzed into α2 and δ subunits, which remain associated as a complex. In this study, we have identified ADAM17 as a key protease involved in proteolytic processing of pro-α2δ-1 and α2δ-3 subunits. We provide three lines of evidence: First, proteolytic cleavage is inhibited by chemical inhibitors of particular metalloproteases, including ADAM17. Second, proteolytic cleavage of both α2δ-1 and α2δ-3 is markedly reduced in cell lines by knockout of ADAM17 but not ADAM10. Third, proteolytic cleavage is reduced by the N-terminal active domain of TIMP-3 (N-TIMP-3), which selectively inhibits ADAM17. We have found previously that proteolytic cleavage into mature α2δ is essential for the enhancement of CaV function, and in agreement, knockout of ADAM17 inhibited the ability of α2δ-1 to enhance both CaV2.2 and CaV1.2 calcium currents. Finally, our data also indicate that the main site of proteolytic cleavage of α2δ-1 is the Golgi apparatus, although cleavage may also occur at the plasma membrane. Thus, our study identifies ADAM17 as a key protease required for proteolytic maturation of α2δ-1 and α2δ-3, and thus a potential drug target in neuropathic pain.

Intracerebral hemorrhage alters α2δ1 and thrombospondin expression in rats

Calcium voltage-gated channel auxiliary subunit (α2δ1) is a non-essential subunit of the voltage-gated calcium channel complex and is ubiquitously expressed in a number of tissues, including the brain. Thrombospondin (TSP)1 and TSP2 are extracellular matrix proteins and belong to the multi-domain glycoprotein family of macromolecular oligomers. TSP1/2 and α2δ1 promote synapse formation and functional recovery following cerebral infarction. However, to the best of our knowledge, the expression levels of α2δ1 and TSP1/2 in brain tissues, and the effects of intracerebral hemorrhage (ICH) on these levels have not yet been elucidated. The present study established a rat model of hemorrhage induced by injecting collagenase IV into the striatum to determine the changes in α2δ1 and TSP1/2 expression following ICH. The protein expression levels of α2δ1 and TSP1 in the striatum after hemorrhage were significantly increased on day 5 and returned to baseline levels on day 21; however, the protein expression levels of TSP2 were decreased on day 5, whereas they increased on day 14, subsequently returning to baseline levels. In addition, using proteomics analysis of tissues from the sham group (saline injection) and at 24 h post-ICH, it was found that both α2δ1 and TSP1 interacted with neural EGFL like 2. Taken together, these findings suggested that the expression levels of α2δ1 and TSP1/2 were altered in brain tissues in response to ICH.

Gabapentinoid-induced peripheral edema and acute heart failure: A translational study combining pharmacovigilance data and in vitro animal experiments

IntroductionGabapentinoids are ligands of the α2-δ subunit of voltage-gated calcium channels (Cav) that have been associated with a risk of peripheral edema and acute heart failure in connection with a potentially dual mechanism, vascular and cardiac. Objectives & methodsAll cases of peripheral edema or heart failure involving gabapentin or pregabalin reported to the French Pharmacovigilance Centers between January 1, 1994 and April 30, 2020 were included to describe their onset patterns (e.g., time to onset). Based on these data, we investigated the impact of gabapentinoids on the myogenic tone of rat third-order mesenteric arteries and on the electrophysiological properties of rat ventricular cardiomyocytes. ResultsA total of 58 reports were included (gabapentin n = 5, pregabalin n = 53). The female-to-male ratio was 4:1 and the median age was 77 years (IQR 57–85, range 32–95). The median time to onset were 23 days (IQR 10–54) and 17 days (IQR 3–30) for non-cardiogenic edema and acute heart failure, respectively. Cardiogenic and non-cardiogenic peripheral edema occurred frequently after a dose escalation (27/45, 60%), and the course was rapidly favorable after discontinuation of gabapentinoid (median 7 days, IQR 5–13). On rat mesenteric arteries, gabapentinoids significantly decreased the myogenic tone to the same extent as verapamil and nifedipine. Acute application of gabapentinoids had no significant effect on Cav1.2 currents of ventricular cardiomyocytes. ConclusionGabapentinoids can cause concentration-dependent peripheral edema of early onset. The primary mechanism of non-cardiogenic peripheral edema is vasodilatory edema secondary to altered myogenic tone, independent of Cav1.2 blockade under the experimental conditions tested.

Calcium current modulation by the γ1 subunit depends on alternative splicing of Cav1.1

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as voltage sensor for excitation-contraction coupling. Conversely, its channel function is limited by multiple mechanisms within the pore-forming α1S subunit and the auxiliary α2δ-1 and γ1 subunits. Particularly, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage-dependence of activation to positive potentials outside the physiological range.

Serum calcium channel subunit α2δ-1 concentrations and outcomes in patients with acute spontaneous intracerebral hemorrhage

BackgroundVoltage-gated calcium channel subunit α2δ-1 plays an important role in acute brain injury. We attempted to investigate whether serum α2δ-1 subunit concentrations are correlated with severity and prognosis following intracerebral hemorrhage (ICH). MethodsSerum α2δ-1 subunit concentrations were quantified in 103 ICH patients and 103 healthy controls. National Institutes of Health Stroke Scale (NIHSS) score and hematoma volume were estimated for assessing illness severity. Modified Rankin scale score of 3–6 at 90 days after stroke onset was defined as a worse outcome. ResultsSerum α2δ-1 subunit concentrations were markedly higher in patients than in controls (median, 875.1 vs. 209.3 pg/ml). Serum α2δ-1 subunit concentrations of patients were tightly correlated with NIHSS score (r = 0.589) and hematoma volume (r = 0.594). Serum α2δ-1 subunit concentrations ≥ 875.1 pg/ml independently discriminated development of 90-day poor outcome with odds ratio of 5.228 (95% CI, 2.201–12.418) and area under the receiver operating characteristic curve of 0.794 (95% CI, 0.703–0.867). Serum α2δ-1 subunit concentrations > 973.4 pg/ml predicted 90-day poor outcome with 64.0% sensitivity and 90.6% specificity. The prognostic predictive ability of serum α2δ-1 concentrations was equivalent to those of NIHSS score and hematoma volume (both P > 0.05), and serum α2δ-1 concentrations also significantly improved the prognostic predictive capabilities of NIHSS score and hematoma volume (both P < 0.05). ConclusionsSerum α2δ-1 subunit concentrations are intimately correlated with illness severity and are independently associated with poor 90-day outcome, substantializing serum α2δ-1 subunit as a potential prognostic biomarker for ICH.

Mirogabalin as a Novel Gabapentinoid for the Treatment of Chronic Pain Conditions: An Analysis of Current Evidence.

Neuropathic pain is a challenge for physicians to treat and often requires a multimodal approach with both pharmacologic and lifestyle interventions. Mirogabalin, a potent, selective ligand of the α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels (VGCCs), provides analgesia by inhibiting neurotransmitter release at the presynaptic end of the neuron. Mirogabalin offers more sustained analgesia than its gabapentinoid counterparts in addition to a wider safety margin for adverse events. Recent clinical trials of mirogabalin have demonstrated both efficacy and tolerability of the drug for the treatment of diabetic peripheral neuropathic pain and postherpetic neuralgia, leading to its approval in Japan. While still not yet FDA approved, mirogabalin is still in its infancy and offers potential into the treatment of neuropathic pain and its associated comorbidities.

Alterations in Excitatory and Inhibitory Synaptic Development Within the Mesolimbic Dopamine Pathway in a Mouse Model of Prenatal Drug Exposure.

The rise in rates of opioid abuse in recent years in the United States has led to a dramatic increase in the incidence of neonatal abstinence syndrome (NAS). Despite improved understanding of NAS and its acute symptoms, there remains a paucity of information regarding the long-term effects of prenatal exposure to drugs of abuse on neurological development. The primary goal of this study was to investigate the effects of prenatal drug exposure on synaptic connectivity within brain regions associated with the mesolimbic dopamine pathway, the primary reward pathway associated with drug abuse and addiction, in a mouse model. Our secondary goal was to examine the role of the Ca+2 channel subunit α2δ-1, known to be involved in key developmental synaptogenic pathways, in mediating these effects. Pregnant mouse dams were treated orally with either the opioid drug buprenorphine (commonly used in medication-assisted treatment for substance use patients), gabapentin (neuropathic pain drug that binds to α2δ-1 and has been increasingly co-abused with opioids), a combination of both drugs, or vehicle daily from gestational day 6 until postnatal day 11. Confocal fluorescence immunohistochemistry (IHC) imaging of the brains of the resulting wild-type (WT) pups at postnatal day 21 revealed a number of significant alterations in excitatory and inhibitory synaptic populations within the anterior cingulate cortex (ACC), nucleus accumbens (NAC), and medial prefrontal cortex (PFC), particularly in the buprenorphine or combinatorial buprenorphine/gabapentin groups. Furthermore, we observed several drug- and region-specific differences in synaptic connectivity between WT and α2δ-1 haploinsufficient mice, indicating that critical α2δ-1-associated synaptogenic pathways are disrupted with early life drug exposure.

Expression, purification and characterization of anti-α2δ1/NanoLuc fusion protein

The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG® M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.

Piperazinyl Bicyclic Derivatives as Selective Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels.

The synthesis and pharmacological activities of a new series of piperazinyl quinazolin-4-(3H)-one derivatives acting toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) are reported. Different positions of a micromolar HTS hit were explored, and best activities were obtained for compounds containing a small alkyl group in position 3 of the quinazolin-4-(3H)-one scaffold and a 3-methyl-piperazin-1-yl- or 3,5-dimethyl-piperazin-1-yl-butyl group in position 2. The activity was shown to reside in the R enantiomer of the chain in position 2, and several eutomers reached single digit nanomolar affinities. Final modification of the central scaffold to reduce lipophilicity provided the pyrido[4,3-d]pyrimidin-4(3H)-one 16RR, which showed high selectivity for Cavα2δ-1 versus Cavα2δ-2, probably linked to its improved analgesic efficacy-safety ratio in mice over pregabalin.

Reader Response: What Is the Mechanism of Therapeutic and Adverse Effects of Gabapentinoids?

Dr. Benarroch's article about gabapentinoids describes that the α2δ subunit of voltage-gated calcium channels (VGCC) allows for neuronal calcium influx. A2δ is expressed in Purkinje cells, which use gamma aminobutyric acid (GABA) as a neurotransmitter. The author states that gabapentinoids inhibit the α2δ subunit and reduce neurotransmitter release, resulting in the induction of cerebellar ataxia.1,2

Editors' Note: What Is the Mechanism of Therapeutic and Adverse Effects of Gabapentinoids?

Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders. Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders.

The Calcium Channel α2δ1 Subunit: Interactional Targets in Primary Sensory Neurons and Role in Neuropathic Pain.

Neuropathic pain is mainly triggered after nerve injury and associated with plasticity of the nociceptive pathway in primary sensory neurons. Currently, the treatment remains a challenge. In order to identify specific therapeutic targets, it is necessary to clarify the underlying mechanisms of neuropathic pain. It is well established that primary sensory neuron sensitization (peripheral sensitization) is one of the main components of neuropathic pain. Calcium channels act as key mediators in peripheral sensitization. As the target of gabapentin, the calcium channel subunit α2δ1 (Cavα2δ1) is a potential entry point in neuropathic pain research. Numerous studies have demonstrated that the upstream and downstream targets of Cavα2δ1 of the peripheral primary neurons, including thrombospondins, N-methyl-D-aspartate receptors, transient receptor potential ankyrin 1 (TRPA1), transient receptor potential vanilloid family 1 (TRPV1), and protein kinase C (PKC), are involved in neuropathic pain. Thus, we reviewed and discussed the role of Cavα2δ1 and the associated signaling axis in neuropathic pain conditions.

Voltage-gated calcium channels (Ca&lt;sub&gt;V&lt;/sub&gt;) in GtoPdb v.2021.3

Ca2+ channels are voltage-gated ion channels present in the membrane of most excitable cells. The nomenclature for Ca2+channels was proposed by [127] and approved by the NC-IUPHAR Subcommittee on Ca2+ channels [70]. Most Ca2+ channels form hetero-oligomeric complexes. The α1 subunit is pore-forming and provides the binding site(s) for practically all agonists and antagonists. The 10 cloned α1-subunits can be grouped into three families: (1) the high-voltage activated dihydropyridine-sensitive (L-type, CaV1.x) channels; (2) the high- to moderate-voltage activated dihydropyridine-insensitive (CaV2.x) channels and (3) the low-voltage-activated (T-type, CaV3.x) channels. Each α1 subunit has four homologous repeats (I-IV), each repeat having six transmembrane domains (S1-S6) and a pore-forming region between S5 and S6. Voltage-dependent gating is driven by the membrane spanning S4 segment, which contains highly conserved positive charges that respond to changes in membrane potential. All of the α1-subunit genes give rise to alternatively spliced products. At least for high-voltage activated channels, it is likely that native channels comprise co-assemblies of α1, β and α2-δ subunits. The γ subunits have not been proven to associate with channels other than the α1s skeletal muscle Cav1.1 channel. The α2-δ1 and α2-δ2 subunits bind gabapentin and pregabalin.

A review of a new voltage-gated Ca2+ channel α2δ ligand, mirogabalin, for the treatment of peripheral neuropathic pain

ABSTRACT Introduction Neuropathic pain (NeP) is a chronic and refractory condition in many patients, and its treatment is a challenge for physicians. A new voltage-gated Ca2+ channel α2δ ligand, mirogabalin, has a high specific binding affinity for the α2δ subunit, with a slower dissociation rate for α2δ-1 than α2δ-2 compared to that of pregabalin. Mirogabalin was shown to be effective in NeP animal models, with a margin of safety between central nervous system side effects and the analgesic effect of the dose. It exerted a favorable analgesic effect, was well tolerated in patients with peripheral NeP (P-NeP), and was first approved in Japan in 2019 and subsequently in Korea and Taiwan in 2020. Areas covered The purpose of this article is to review the pharmacological characteristics, pharmacokinetics, and efficacy and safety of mirogabalin for NeP based on the results of non-clinical and clinical studies. Expert opinion Although there are several first-line therapies for NeP, insufficient efficacy and adverse drug reactions of NeP drugs often cause patient dissatisfaction. Mirogabalin was effective and well tolerated with a step-wise dose increase in clinical studies on P-NeP patients. Thus, mirogabalin is expected to be a useful treatment option for patients with P-NeP.

Input-dependent synaptic suppression by pregabalin in the central amygdala in male mice with inflammatory pain

Pregabalin (PGB) is a synthetic amino acid compound most widely prescribed for chronic peripheral and central neuropathic pain. PGB is a ligand for the α2δ1 subunit of voltage-dependent calcium channels, and its binding reduces neurotransmitter release and thus inhibits synaptic transmission. The central nucleus of the amygdala (CeA) is a kernel site for the enhanced nociception-emotion link in chronic pain. The nociceptive information is conveyed to the CeA via the following two pathways: 1) the pathway arising from the basolateral amygdala (BLA), which carries nociceptive information mediated by the thalamocortical system, and 2) that arising from the external part of the pontine lateral parabrachial nucleus (LPB), that forms the final route of the spino-parabrachio-amygdaloid pathway that conveys nociceptive information directly from the superficial layer of the spinal dorsal horn. We compared the effects of PGB on the excitatory postsynaptic currents of neurons in the right CeA in response to electrical stimulation of BLA and LPB pathways using the whole-cell patch-clamp technique. Inflammatory pain was induced by intraplantar injection of formalin solution at the left hind paw.At eight hours post-formalin, PGB reduced EPSCs amplitude of the BLA-to-CeA synaptic transmission, accompanied by a significant increase in the PPR, suggesting a decreased release probability from the presynaptic terminals. In addition, these effects of PGB were only seen in inflammatory conditions. PGB did not affect the synaptic transmission at the LPB-to-CeA pathway, even in formalin-treated mice. These results suggest PGB improves not simply the aberrantly enhanced nociception but also various pain-associated cognitive and affective consequences in patients with chronic nociplastic pain.

Structure of human Cav2.2 channel blocked by the painkiller ziconotide.

The neuronal-type (N-type) voltage-gated calcium (Cav) channels, which are designated Cav2.2, have an important role in the release of neurotransmitters1-3. Ziconotide is a Cav2.2-specific peptide pore blocker that has been clinically used for treating intractable pain4-6. Here we present cryo-electron microscopy structures of human Cav2.2 (comprising the core α1 and the ancillary α2δ-1 and β3 subunits) in the presence or absence of ziconotide. Ziconotide is thoroughly coordinated by helices P1 and P2, which support the selectivity filter, and the extracellular loops (ECLs) in repeats II, III and IV of α1. To accommodate ziconotide, the ECL of repeat III and α2δ-1 have to tilt upward concertedly. Three of the voltage-sensing domains (VSDs) are in a depolarized state, whereas the VSD of repeat II exhibits a down conformation that is stabilized by Cav2-unique intracellular segments and a phosphatidylinositol 4,5-bisphosphate molecule. Our studies reveal the molecular basis for Cav2.2-specific pore blocking by ziconotide and establish the framework for investigating electromechanical coupling in Cav channels.

Voltage-gated calcium channels in GtoPdb v.2021.2

Calcium (Ca2+) channels are voltage-gated ion channels present in the membrane of most excitable cells. The nomenclature for Ca2+channels was proposed by [127] and approved by the NC-IUPHAR Subcommittee on Ca2+ channels [70]. Most Ca2+ channels form hetero-oligomeric complexes. The α1 subunit is pore-forming and provides the binding site(s) for practically all agonists and antagonists. The 10 cloned α1-subunits can be grouped into three families: (1) the high-voltage activated dihydropyridine-sensitive (L-type, CaV1.x) channels; (2) the high- to moderate-voltage activated dihydropyridine-insensitive (CaV2.x) channels and (3) the low-voltage-activated (T-type, CaV3.x) channels. Each α1 subunit has four homologous repeats (I-IV), each repeat having six transmembrane domains and a pore-forming region between transmembrane domains S5 and S6. Voltage-dependent gating is driven by the membrane spanning S4 segment, which contains highly conserved positive charges that respond to changes in membrane potential. All of the α1-subunit genes give rise to alternatively spliced products. At least for high-voltage activated channels, it is likely that native channels comprise co-assemblies of α1, β and α2-δ subunits. The γ subunits have not been proven to associate with channels other than the α1s skeletal muscle Cav1.1 channel. The α2-δ1 and α2-δ2 subunits bind gabapentin and pregabalin.

Calcium Channel Subunit α2δ-1 as a Potential Biomarker Reflecting Illness Severity and Neuroinflammation in Patients with Acute Ischemic Stroke

BackgroundVoltage-gated calcium channels (VGCCs) dysfunction is involved in the development of acute ischemic stroke (AIS). As a subunit of VGCC complexes, we detected the levels of α2δ-1 subunit in serum and cerebrospinal fluid (CSF) specimens from AIS patients. MethodsThe study included 105 patients with first-ever AIS, who were admitted within 48 hours after stroke onset. The serum and CSF levels of α2δ-1 were measured with ELISA and the severity of AIS patients was evaluated according to the National Institutes of Health Stroke Scale (NIHSS) score. The cerebral infarct volume was calculated through the Pullicino formula based on the cranial CT or MRI scan. C-reactive protein (CRP) and serum amyloid A (SAA) were measured using the latex-enhanced immunoturbidimetric assay. ResultsCompared to the control subjects, the serum α2δ-1 level was significantly increased in AIS patients with large infarct volume and in severe AIS cases with high NIHSS score, which correlated positively with the inflammatory markers CRP and SAA. Furthermore, the concentration of α2δ-1 in CSF was elevated with the infarct volume, which was higher in severe AIS patients. ConclusionOur study suggests that the increased α2δ-1 levels in serum and CSF specimens may be used as a potential marker for reflecting VGCCs dysfunction, illness severity and neuroinflammation in AIS patients.