Analgesic alpha-Conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABA-B receptor activation

alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha 9 alpha 10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P60] Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the alpha-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDP beta S, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABAB receptor antagonists, [S-(R*, R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify CaV2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABAB receptors

Functional correlates of clinical phenotype and severity in recurrent SCN2A variants

In SCN2A-related disorders, there is an urgent demand to establish efficient methods for determining the gain- (GoF) or loss-of-function (LoF) character of variants, to identify suitable candidates for precision therapies. Here we classify clinical phenotypes of 179 individuals with 38 recurrent SCN2A variants as early-infantile or later-onset epilepsy, or intellectual disability/autism spectrum disorder (ID/ASD) and assess the functional impact of 13 variants using dynamic action potential clamp (DAPC) and voltage clamp. Results show that 36/38 variants are associated with only one phenotypic group (30 early-infantile, 5 later-onset, 1 ID/ASD). Unexpectedly, we revealed major differences in outcome severity between individuals with the same variant for 40% of early-infantile variants studied. DAPC was superior to voltage clamp in predicting the impact of mutations on neuronal excitability and confirmed GoF produces early-infantile phenotypes and LoF later-onset phenotypes. For one early-infantile variant, the co-expression of the alpha(1) and beta(2) subunits of the Na(v)1.2 channel was needed to unveil functional impact, confirming the prediction of 3D molecular modeling. Neither DAPC nor voltage clamp reliably predicted phenotypic severity of early-infantile variants. Genotype, phenotypic group and DAPC are accurate predictors of the biophysical impact of SCN2A variants, but other approaches are needed to predict severity. A comprehensive biophysical analysis of disease-associated mutations in the voltage-gated sodium channel gene, SCN2A, suggests that dynamic action potential clamp may be a better predictor than voltage clamp of how these mutations alter neuronal excitability, though other approaches are needed to predict severity

Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.Peer reviewe

Dietary fish oil reduces the occurrence of early afterdepolarizations in pig ventricular myocytes

Fish oil reduces sudden cardiac death in post myocardial infarction patients. Life-threatening arrhythmias in heart failure are associated with repolarization abnormalities leading to EAD(1) formation. We examined the effects of incorporated fish oil omega 3-PUFAs2 on EAD formation in pig myocytes. Pigs were fed a diet rich in fish oil or sunflower oil (control) for 8 weeks. Myocytes were isolated by enzymatic dissociation and patch-clamped. Susceptibility to EAD formation was tested using E4031 (5 mu M), a blocker of I-Kr. The fish oil diet in pigs resulted in increased incorporation of omega 3-PUFAs in the sarcolemma of the myocytes compared to the control diet and caused a reduced occurrence of E4031-induced EADs in pig myocytes. A shorter action potential, a reduced action potential prolongation in response to E-4031 and a reduced reactivation of I-Ca,I-L by omega 3-PUFAs may explain the observed reduction in EADs. A diet rich in fish oil protects against EAD formation. (c) 2006 Elsevier Inc. All rights reserved

Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1

Cyclic alpha-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H, C8F] cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human alpha 9 alpha 10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 mu M and rat N-type (Ca(v)2.2) and recombinant human Ca(v)2.3 calcium channels via GABA(B) receptor activation, with an IC50 of similar to 900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis

Low Voltage Activation of KCa1.1 Current by Cav3-KCa1.1 Complexes

<div><p></p><p>Calcium-activated potassium channels of the KCa1.1 class are known to regulate repolarization of action potential discharge through a molecular association with high voltage-activated calcium channels. The current study examined the potential for low voltage-activated Cav3 (T-type) calcium channels to interact with KCa1.1 when expressed in tsA-201 cells and in rat medial vestibular neurons (MVN) <i>in vitro</i>. Expression of the channel α-subunits alone in tsA-201 cells was sufficient to enable Cav3 activation of KCa1.1 current. Cav3 calcium influx induced a 50 mV negative shift in KCa1.1 voltage for activation, an interaction that was blocked by Cav3 or KCa1.1 channel blockers, or high internal EGTA. Cav3 and KCa1.1 channels coimmunoprecipitated from lysates of either tsA-201 cells or rat brain, with Cav3 channels associating with the transmembrane S0 segment of the KCa1.1 N-terminus. KCa1.1 channel activation was closely aligned with Cav3 calcium conductance in that KCa1.1 current shared the same low voltage dependence of Cav3 activation, and was blocked by voltage-dependent inactivation of Cav3 channels or by coexpressing a non calcium-conducting Cav3 channel pore mutant. The Cav3-KCa1.1 interaction was found to function highly effectively in a subset of MVN neurons by activating near –50 mV to contribute to spike repolarization and gain of firing. Modelling data indicate that multiple neighboring Cav3-KCa1.1 complexes must act cooperatively to raise calcium to sufficiently high levels to permit KCa1.1 activation. Together the results identify a novel Cav3-KCa1.1 signaling complex where Cav3-mediated calcium entry enables KCa1.1 activation over a wide range of membrane potentials according to the unique voltage profile of Cav3 calcium channels, greatly extending the roles for KCa1.1 potassium channels in controlling membrane excitability.</p></div

Cav3.2 and KCa1.1 channels associate in rat brain.

<p><i>A,</i> Dual label immunocytochemistry confirms Cav3.2 and KCa1.1 protein expression in MVN cells with a similar distribution pattern at the level of the soma and at least the proximal dendritic region. <i>B,</i> Control image upon omission of primary antibodies. <i>C,</i> Western blots showing coimmunoprecipitation of Cav3.2 and KCa1.1 protein from lysates of rat brain (<i>n</i> = 4), cerebellum (<i>n</i> = 6), and brain stem (<i>n</i> = 5), with a corresponding label for KCa1.1 in lysates from each region. In lane 1, channel complexes were immunoprecipitated using the Cav3.2 antibody, in lane 2 the precipitating antibody was omitted, and lane 3 corresponds to lysate. All Western blots were probed with anti-KCa1.1. Scale bar in (<i>A, B</i>) = 20 µm.</p

Ca_v3.2 channels interact with the N-terminal transmembrane region of KCa1.1 channels.

<p><i>A</i>, A schematic drawing of the α-subunit of KCa1.1 with the N-terminal segment. Two alternate N-terminal splice variant sequences are shown below the diagram (N+S0, aN+S0) with S0 designating the transmembrane component of the N-terminal region. Region of overlap between KCa1.1 N+S0 and aN+S0 are shown in <i>grey shading</i>. Constructs tested include full length KCa1.1, KCa1.1 N+S0, KCa1.1 aN+S0, KCa1.1 N-terminus, KCa1.1 without C-terminus (KCa1.1ΔC-term), and KCa1.1 C-terminus (<i>n</i> = 4). <i>B</i>, <i>Left column</i>, Western blots from lysates of tSA-201 cells indicating that Ca_v3.2 protein coimmunoprecipitates with full length KCa1.1, KCa1.1ΔC-term, or KCa1.1 N+S0. <i>Right column</i>, Cav3.2 channels coimmunoprecipitate with KCa1.1 aN+S0 but not with the KCa1.1 N-terminus. A weak binding was also observed with KCa1.1 C-terminus and Cav3.2. In both panels B and C the first lane corresponds to an immunoprecipitation conducted with a polyclonal Cav3.2 antibody. In the second lane, the precipitating antibody was omitted (i.e., bead only control), and the third lane corresponds to tsA-201 cell lysate. In each case, Western blots were probed with an anti-myc antibody to detect myc-tagged KCa1.1 channels or their fragments. Sample sizes are shown in brackets.</p