The Brugada syndrome mutation A39V does not affect surface expression of neuronal rat Cav1.2 channels

<p>Abstract</p> <p>Background</p> <p>A loss of function of the L-type calcium channel, Cav1.2, results in a cardiac specific disease known as Brugada syndrome. Although many Brugada syndrome channelopathies reduce channel function, one point mutation in the N-terminus of Cav1.2 (A39V) has been shown to elicit disease a phenotype because of a loss of surface trafficking of the channel. This lack of cell membrane expression could not be rescued by the trafficking chaperone Cavβ.</p> <p>Findings</p> <p>We report that despite the striking loss of trafficking described previously in the cardiac Cav1.2 channel, the A39V mutation while in the background of the brain isoform traffics and functions normally. We detected no differences in biophysical properties between wild type Cav1.2 and A39V-Cav1.2 in the presence of either a cardiac (Cavβ2b), or a neuronal beta subunit (Cavβ1b). In addition, the A39V-Cav1.2 mutant showed a normal Cavβ2b mediated increase in surface expression in tsA-201 cells.</p> <p>Conclusions</p> <p>The Brugada syndrome mutation A39V when introduced into rat brain Cav1.2 does not trigger the loss-of-trafficking phenotype seen in a previous study on the human heart isoform of the channel.</p

Effect of the Brugada syndrome mutation A39V on calmodulin regulation of Cav1.2 channels

BACKGROUND: The L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (N-lobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels. RESULTS: We examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the N- and/or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process. CONCLUSIONS: Our data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating

Pathogenic Cav3.2 channel mutation in a child with primary generalized epilepsy.

Two paternally-inherited missense variants in CACNA1H were identified and characterized in a 6-year-old child with generalized epilepsy. Febrile and unprovoked seizures were present in this child. Both variants were expressed in cis or isolation using human recombinant Cav3.2 calcium channels in tsA-201 cells. Whole-cell patch-clamp recordings indicated that one variant (c.3844C &gt; T; p.R1282W) caused a significant increase in current density consistent with a pathogenic gain-of-function phenotype; while the other cis-related variant (c.5294C &gt; T; p.A1765V) had a benign profile

Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition

Direct interaction with the β subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel α1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gβ1γ2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the α1 subunit, residues which are surface-exposed in the presence of the calcium channel β subunit, contribute significantly to the functional inhibition by Gβ1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gβ1γ2 subunits and with several isoforms of the auxiliary β subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel

Cellular prion protein protects from inflammatory and neuropathic pain

Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours

Cysteine String Protein Regulates G Protein Modulation of N-Type Calcium Channels

AbstractCysteine string proteins (CSPs) are secretory vesicle proteins bearing a “J domain” and a palmitoylated cysteine-rich “string” region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. Gα subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gβγ subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission

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

Background: Cannabinoid 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. Results: NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTP gamma[S-35] 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 Ca(V)3.2 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 Ca(V)3.2 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). Conclusions: Our 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

NMP-7 inhibits chronic inflammatory and neuropathic pain via block of Cab3.2 T-type calcium channels and activation of CB2 receptors

Background: T-type calcium channels and cannabinoid receptors are known to play important roles in chronic pain, making them attractive therapeutic targets. We recently reported on the design, synthesis and analgesic properties of a novel T-type channel inhibitor (NMP-7), which also shows mixed agonist activity on CB1 and CB2 receptors in vitro. Here, we analyzed the analgesic effect of systemically delivered NMP-7 (intraperitoneal (i.p.) or intragstric (i.g.) routes) on mechanical hypersensitivity in inflammatory pain induced by Complete Freund’s Adjuvant (CFA) and neuropathic pain induced by sciatic nerve injury. Results: NMP-7 delivered by either i.p. or i.g. routes produced dose-dependent inhibition of mechanical hyperalgesia in mouse models of inflammatory and neuropathic pain, without altering spontaneous locomotor activity in the open-field test at the highest active dose. Neither i.p. nor i.g. treatment reduced peripheral inflammation per se, as evaluated by examining paw edema and myeloperoxidase activity. The antinociception produced by NMP-7 in the CFA test was completely abolished in CaV3.2-null mice, confirming CaV3.2 as a key target. The analgesic action of intraperitoneally delivered NMP-7 was not affected by pretreatment of mice with the CB1 antagonist AM281, but was significantly attenuated by pretreatment with the CB2 antagonist AM630, suggesting that CB2 receptors, but not CB1 receptors are involved in the action of NMP-7 in vivo. Conclusions: Overall, our work shows that NMP-7 mediates a significant analgesic effect in a model of persistent inflammatory and chronic neuropathic pain by way of T-type channel modulation and CB2 receptor activation. Thus, this study provides a novel therapeutic avenue for managing chronic pain conditions via mixed CB ligands/ T-type channel blockers

Differential Interactions of Na+ Channel Toxins with T-type Ca2+ Channels

Two types of voltage-dependent Ca2+ channels have been identified in heart: high (ICaL) and low (ICaT) voltage-activated Ca2+ channels. In guinea pig ventricular myocytes, low voltage–activated inward current consists of ICaT and a tetrodotoxin (TTX)-sensitive ICa component (ICa(TTX)). In this study, we reexamined the nature of low-threshold ICa in dog atrium, as well as whether it is affected by Na+ channel toxins. Ca2+ currents were recorded using the whole-cell patch clamp technique. In the absence of external Na+, a transient inward current activated near −50 mV, peaked at −30 mV, and reversed around +40 mV (HP = −90 mV). It was unaffected by 30 μM TTX or micromolar concentrations of external Na+, but was inhibited by 50 μM Ni2+ (by ∼90%) or 5 μM mibefradil (by ∼50%), consistent with the reported properties of ICaT. Addition of 30 μM TTX in the presence of Ni2+ increased the current approximately fourfold (41% of control), and shifted the dose–response curve of Ni2+ block to the right (IC50 from 7.6 to 30 μM). Saxitoxin (STX) at 1 μM abolished the current left in 50 μM Ni2+. In the absence of Ni2+, STX potently blocked ICaT (EC50 = 185 nM) and modestly reduced ICaL (EC50 = 1.6 μM). While TTX produced no direct effect on ICaT elicited by expression of hCaV3.1 and hCaV3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni2+ (IC50 increased to 550 μM Ni2+ for CaV3.1 and 15 μM Ni2+ for CaV3.2); in contrast, 30 μM TTX directly inhibited hCaV3.3-induced ICaT and the addition of 750 μM Ni2+ to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni2+ alone. 1 μM STX directly inhibited CaV3.1-, CaV3.2-, and CaV3.3-mediated ICaT but did not enhance the ability of Ni2+ to block these currents. These findings provide important new implications for our understanding of structure–function relationships of ICaT in heart, and further extend the hypothesis of a parallel evolution of Na+ and Ca2+ channels from an ancestor with common structural motifs