Human Cav3 Research Articles

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel.

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

Dicarba analogues of α-conotoxin RgIA. Structure, stability, and activity at potential pain targets.

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors.

Association between upstream purine complexes of human caveolin-1 gene and schizophrenia in qazvin province of iran.

Background:Caveolin is a multifunctional and scaffolding membrane protein, which involves cholesterol trafficking to plasma lipid microdomain. It organizes and targets synaptic parts of the neurotransmitter and neurotrophic receptor signaling pathways. Caveolins are encoded by CAV-1, 2 and 3 genes. Disruption of the CAV1 would likely ruin the neuronal signaling, which leads to symptoms of schizophrenia in predisposed individuals.Objectives:The upper area of CAV-1 gene is highly conserved and can have a regulatory role in neurodegenerative diseases. This study was designed to find out the possible association of polymorphisms of this area and schizophrenia.Patients and Methods:In a case-control study, 254 blood samples were obtained from 127 patients with schizophrenia and 127 well matched controls referred to 22 Bahman Hospital of Qazvin University of Medical Sciences (QUMS) in Qazvin province, Iran, using simple random sampling method. After extracting DNA, the upper region of the human CAV1- gene was amplified by PCR in all collected samples. The products were visualized by silver staining in 10% polyacrylamide gel and then sequenced.Results:We detected nine homozygotes in patients and 15 in control subjects. Homozygosity was 7.08% and 11.8% in cases and control, respectively. Nine types homozygote haplotype were detected in upper region of the CAV1 gene in cases and controls. Three haplotypes were common in cases and controls; four haplotypes were seen in controls only and two in cases.Conclusions:Our findings implied a significant correlation between some haplotypes of upper region of CAV1 gene and schizophrenia. Existence of some haplotypes and lack of another in CAV1 upstream can suggest a significant correlation between schizophrenia and some haplotypes.

GW25-e4410 The Effects of Lysophosphatidylcholine on action potentials of cardiomyocyets and its mechanisms

To explore the effects of LPC on action potentials of cardiomyocytes and its mechanisms. Neonatal rat cardiomyocytes from 1 to 3-days Wistar rats were prepared, and the specific Human cardiac T-type calcium channel a1 subunits, Cav3.1 and Cav3.2, were stably expressed in HEK293 cells (HEK293-Cav3.1

Modest effects of lipid modifications on the structure of caveolin-3.

Caveolin-3 (Cav3) is an unconventional membrane protein that serves as a critical scaffolding hub in caveolae and is genetically linked to various muscle disorders. In this work, we report the expression, purification, and characterization of full-length human Cav3. To mimic the palmitoylation of endogenous Cav3, we developed a generally applicable approach to covalently attached thioalkyl chains at natively modified cysteine residues. Nuclear magnetic resonance measurements indicate that lipidation exerts only a modest and local effect on the Cav3 structure, with little impact on the structures of the N-terminal domain, the scaffolding domain, and the extreme C-terminus.

Identifying and predicting the impact of functional single nucleotide polymorphisms (SNPs) in human CAV-3 gene

Genetic mutations, such as Single nucleotide polymorphisms (SNPs) are known to be the most reported type of genetic variations in individuals and thought to have a crucial impact on the understanding of many genetic related diseases. One of the major challenges is to find the deleterious SNPs in disease-associated genes. In the current study, computational methods were used to analyze the SNPs which may effect the expression and function of CAV 3 gene. Of the total 377 SNPs, 288(76.8%) were found to be non-coding SNPs, while 53 (14.1%) were non synonymous (ns)SNPs and 4(1.1%) were in the mRNA UTRs. Forty nine nsSNPs encoding missense mutation were tested for the parable effect on protein structure and functions using SIFT, I-Mutant 2, PolyPhen 2 and PANTHER soft wares. Moreover, one nsSNP (R27Q) was further analyzed using HOPE project, and this mutation predicted to substantially affect the structure along with the function of caveolin 3 protein.

Human CaV1.2/CaV3.x channels mediate paradoxical vasomotor responses in the human cerebral circulation (677.11)

This study examined which voltage‐gated Ca2+ channels are expressed in the human cerebral circulation and investigated their electrical and functional properties. Using cerebral arteries collected from patients undergoing resection surgeries, quantitative PCR and Western blot analyses revealed the expression of both L‐ and T‐type channels in the smooth muscle cell layer. Analogous to rodent, we observed both CaV1.2 (L) and CaV3.2 (T); intriguingly, CaV3.1 isoform was replaced in humans with CaV3.3 (T). Patch‐clamp electrophysiology displayed a robust whole‐cell Ba2+ current whose amplitude varied inversely with the patients’ age. This inward current was subdivided into L‐ and T‐type components using standard pharmacological/electrical approaches. The T‐type conductance was further separated into CaV3.2 and CaV3.3 components by exploiting their differential sensitivity to Ni2+. Vessel myography revealed a key role for CaV1.2 and CaV3.3 channels in mediating cerebral arterial constriction with the former and latter predominating at depolarized and hyperpolarized voltages, respectively. Like the rodent, human CaV3.2 appears to drive a negative feedback response that antagonizes myogenic tone development. In conclusion, this study is the first to document three distinct Ca2+ channel isoforms in human cerebral arteries along with their unique contribution to arterial tone development.

Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation

Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba(2+) currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5'-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.

A Timothy Syndrome Causing Mutation Perturbs Voltage Sensor Operation in Human CaV1.2 Channels

The pore-forming α1C subunit of the voltage-gated L-type calcium channel (CaV1.2) consists of four concatenated Repeats (I-IV), each contributing a voltage-sensing domain (VSD, transmembrane helices S1-S4) and a quarter of the central ion-conductive pore domain (S5-S6). In Timothy Syndrome (TS), a rare and severe multi-organ disorder, specific alterations in CaV1.2 voltage-dependent properties lead to autism, immunodeficiency, QT prolongation (LQT8) and lethal arrhythmias. The most frequent TS-causing mutation (G406R) is located at the intracellular flank of S6 in Repeat I and promotes Ca2+ influx by (1) shifting CaV1.2 voltage- dependent activation towards more negative potentials and (2) impairing voltage-dependent inactivation. To gain insight on the molecular basis underlying the TS-causing voltage-sensitivity anomalies, we studied the structural rearrangements of VSDs I and III in human CaV1.2 channels carrying the G406R mutation, using the voltage-clamp fluorometry technique. Briefly, this involves conjugating a small, environment-sensitive fluorophore at a specific VSD that reports local conformational rearrangements as fluorescence changes. Under voltage-clamp, we simultaneously acquired ionic current and fluorescence signals, which corresponded to specific VSD rearrangements during channel activation. We found that the G406R mutation dramatically altered the operation of VSDs I and III compared to wild-type channels, by inducing a hyperpolarizing shift in their activation voltage dependence of ∼80mV and ∼50mV, respectively. These shifts were associated with a significant reduction in the effective valences of VSD I and III by ∼50% and ∼42%, respectively. Moreover, the sign of the fluorescence signals detected from TS channels was opposite to that observed in wild-type CaV1.2 channels. Taken together, these results suggest that the TS-causing mutation causes an overall structural perturbation, manifested as a change in both the fluorophore quenching process reported from VSD I and VSD III and their altered voltage-dependence. Funded by: NIH,AHA,FONDECYT,ACT.

Differential CaV2.1 and CaV2.3 Channel Inhibition by Baclofen and α-Conotoxin Vc1.1 via GABAB Receptor Activation

In the nervous system, Cav2.1 (P/Q-type), Cav2.2 (N-type) and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated via G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 has been identified as a selective inhibitor of Cav2.2 channels acting via GABAB receptors. We investigated baclofen and Vc1.1 modulation of human Cav2.1 or Cav2.3 channels via human GABAB receptor activation in HEK cells. Baclofen (50 µM) inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by ∼ 40%, whereas Vc1.1 did not affect Cav2.1, but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼ 300 pM. Depolarizing paired-pulses revealed that ∼ 75% of the baclofen inhibition of Cav2.1 was voltage-dependent, and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated via voltage-independent pathways that could be disrupted by pertussis toxin, GDP-β-S or the GABAB receptor antagonist, CGP55845. Over-expression of c-Src kinase significantly increased inhibition of Cav2.3 by Vc1.1. Conversely, co-expression of a double mutant c-Src or pre-treatment with a phosphorylated pp60c-Src peptide abolished the effect of Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for mediating the inhibition by Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conveyed Vc1.1 sensitivity. Our findings demonstrate that Vc1.1 inhibition of Cav2.3 is due to specific c-Src phosphorylation sites in the C-teminus, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins.

The Functional Heterogeneity of the Human CaV1.2 Voltage Sensors

Voltage-dependent Ca2+ channels (CaV) are the principal pathway of excitation-evoked Ca2+ entry. Their pore-forming α1C subunit encodes four homologous, but non-identical, concatenated repeats, each including a voltage-sensing domain (VSD). The four VSDs confer voltage dependence to the ion-conductive pore. A potential consequence of the independent evolution of each VSD is that the strength and timing of Ca2+ influx are governed by up to four VSDs with distinct voltage-sensing properties and contributions to channel opening. To test this hypothesis, and resolve the voltage dependent conformational rearrangements that initiate excitation-coupled Ca2+ signaling, we used voltage-clamp fluorometry, tracking the activation of each VSD in human CaV1.2 (L-type) channels coexpressed with β3 and α2δ subunits. We found that each VSD possesses distinct voltage-sensitivity, as estimated by fitting normalized fluorescence deflections from each VSD to Boltzmann distributions: VSD-I: V0.5= 8.3±2.7mV; z=2.0±0.090e0; VSD-II: V0.5=-27±1.8mV; z=2.4±0.17e0; VSD-III: V0.5=-18±2.1mV; z=1.4±0.085e0; VSD-IV: V0.5=-61±2.3mV; z=1.3±0.18e0. Upon depolarization to 0mV, VSDs I-III activate with a similar time course to channel opening (τON≈2-4ms), while VSD-IV is significantly slower (τON=17±2.3ms). Upon repolarization, VSD-II deactivates with a kinetic component practically synchronous to the tail current (τOFF=0.57±0.22ms), while other VSDs were several fold slower: VSD-I τOFF=3.7±0.69ms; VSD-III τOFF=6.6±1.0ms; VSD-IV τOFF=28±5.3ms. How do the disparate voltage dependences and kinetics of the four VSDs associate with open probability and global charge displacement? All experimental data were fit by an allosteric model comprising five “gating particles” representing the four VSDs and the pore. This model revealed that the pore Closed/Open transition in the α1C-β3-α2δ channel is primarily regulated by VSD-II and -III, which stabilize the Open state by ∼-37meV and ∼-68meV, respectively. This finding sets CaV1.2 channels apart from KV and NaV channels, where activation is governed by four and three VSDs, respectively. Funded by: NIH,AHA,FONDECYT,ACT.

PDZ-domain scaffold proteins differentially modulate neuronal membrane expression of C-terminal splice variants of the human L-type calcium channel CaV1.3

PDZ-domain scaffold proteins differentially modulate neuronal membrane expression of C-terminal splice variants of the human L-type calcium channel CaV1.3

Caveolin-1 Protects B6129 Mice against Helicobacter pylori Gastritis

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies (“humming bird”) compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells.

Expression and Pharmacology of Endogenous Cav Channels in SH-SY5Y Human Neuroblastoma Cells

SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms) and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1–3, β1, β3, β4, γ1, γ4–5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (∼ 10-fold) higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.

The familial hemiplegic migraine type 1 mutation K1336E affects direct G protein-mediated regulation of neuronal P/Q-type Ca2+ channels

Familial hemiplegic migraine type 1 (FHM-1) is an autosomal dominant form of migraine with aura characterized by recurrent migraine, hemiparesis and ataxia. FHM-1 has been linked to missense mutations in the CACNA1A gene encoding the pore-forming subunit of the neuronal voltage-gated P/Q-type Ca(2+) channel (CaV2.1α1). Here, we explored the effects of the FHM-1 K1336E mutation on G protein-dependent modulation of the recombinant P/Q-type channel. The mutation was introduced into the human CaV2.1α1 subunit and its functional consequences investigated after heterologous expression in HEK-293 cells using patch-clamp recordings. Functional analysis of the K1336E mutation revealed a reduction of Ca(2+) current densities, a ∼10 mV left-shift in the current-voltage relationship, and the slowing of current inactivation kinetics. When co-expressed along with the human μ-opioid receptor, application of the agonist DAMGO inhibited whole-cell currents through both the wild-type and the mutant channels. Prepulse facilitation was also reduced by the K1336E mutation. Likewise, the kinetic analysis of the onset and decay of facilitation showed that the mutation affects the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex. These results suggest that the extent of G-protein-mediated inhibition is significantly reduced in the K1336E mutant CaV2.1 Ca(2+) channels. This alteration would contribute to render the neuronal network hyperexcitable, possibly as a consequence of reduced presynaptic inhibition, and may help to explain some aspects of the FHM-1 pathophysiology.

PrP octarepeats region determined the interaction with caveolin-1 and phosphorylation of caveolin-1 and Fyn

Caveolin-1 is one of the major constituents of caveolae. Both Cav-1 and PrP are plasma membrane proteins, which show active capacities for molecular interactions with many other proteins or agents, including themselves. Using yeast two-hybrid system and immunoprecipitation, we reconfirmed the molecular interaction between human Cav-1 and PrP. With co-immunoprecipitation tests, PrP(C)-Cav-1 and PrP(Sc)-Cav-1 complexes were identified in the brain homogenates of normal and scrapie agent 263K-infected hamsters, respectively. Transient expression of wild-type PrP (PrP-PG5) in HEK293 cells did not change the situation of Cav-1 and subsequent signal transduction pathways, while cross-linking of the expressed PrP with specific antibody induced remarkable colocalization of PrP and Cav-1 on the plasma membrane and significant increases of phosphorylated Cav-1 and phosphorylated Fyn. With deleted and inserted PrP mutants within octarepeat region, we observed obvious octarepeat-associated phenomena, including lower binding capacity with Cav-1 in vitro, unable to co-localize with Cav-1 in the cells and to induce up-regulation of p-Cav-1 and p-Fyn when removal of octarepeats in the context of full-length PrP. Moreover, we found that treatment on HEK293 cells with fibrous form of recombinant PrP protein led to up-regulating the levels of p-Cav-1 and p-Fyn. Our data here provide strong evidence that octarepeats of PrP are critical for the interaction between PrP and Cav-1. Significant alterations in the cultured cells, either the distributions of PrP and Cav-1 morphologically or the up-regulations of p-Cav-1 and p-Fyn, induced by antibody-mediated cross-linking or fibrous forms of PrP may suggest a possible internalization process of PrP(Sc).

Optically-Resolved Conformational Rearrangements of the Voltage-Sensing Domains of the Human CaV1.2 Channel

Voltage-gated Ca2+ channels (CaV) consist of four tandem transmembrane repeats (I-IV), each including two pore-forming helices and four segments that make up a voltage-sensing domain (VSD). To understand voltage-dependent gating in human CaV1.2 channels, we have optically tracked the activation of the S4 helices of repeats I, III and IV by site-directed fluorescent labeling of introduced Cysteines with thiol-reactive probes. The channels were co-expressed with their modulatory subunits β3 and α2δ in Xenopus oocytes. Ionic current and fluorescence emission were simultaneously recorded using the cut-open oocyte voltage clamp fluorometry technique. Prior voltage-clamp, oocytes were injected with 100 nl 80 mM BAPTA.4K, to prevent the activation of endogenous Ca2+-gated Cl− channels. The extracellular solution contained 2 mM Ba2+ or 10 mM Ca2+ as charge carrier. Gating currents were recorded by replacing Ca2+ or Ba2+ with Co2+. 0.1 mM ouabain was added to abolish Na+/K+ ATPase non-linear charge movement. Voltage-dependent fluorescence changes (ΔF) were reported by fluorophores attached to substituted Cysteines at the extracelular end of the S4 segment in repeats I, III and IV, tracking local, voltage-dependent conformational rearrangements. The voltage dependence of observed fluorescence deflections preceded ionic activation, reporting repeat-specific VSD transitions taking place during shut states of the channel. Prolonged sojourns at depolarized potentials (+50 mV) shifted the voltage-dependence of reported ΔF to more hyperpolarized potentials by >50mV, recapitulating previously-characterized gating current properties. In summary, we report the first optical characterization of VSD conformational changes in a human voltage-gated Ca2+ channel, revealing repeat-specific voltage- and time-dependent properties.

Analgesic Effect of a Mixed T-Type Channel Inhibitor/CB2 Receptor Agonist

BackgroundCannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181.ResultsNMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTPγ[35S] functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak CaV3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in CaV3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist).ConclusionsOur work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands.

CaV1.2 I-II linker structure and Timothy syndrome

CaV channels are multi-subunit protein complexes that enable inward cellular Ca2+ currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker’s helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the CaV1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant’s (human CaV1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.

The human L-type calcium channel Cav1.3 regulates insulin release and polymorphisms in CACNA1D associate with type 2 diabetes

Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes. Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Cav)1.2 and Cav1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Cav1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes. In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Cav1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes. We conclude that the L-type calcium channel Cav1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.