Differential Effects of RGK Proteins on L-Type Channel Function in Adult Mouse Skeletal Muscle

Work in heterologous systems has revealed that members of the Rad, Rem, Rem2, Gem/Kir (RGK) family of small SIP-binding proteins profoundly inhibit L-type Ca2+ channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of P-o without impaired voltage-sensor movement. However, the question of which mode is the critical one for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L-type Ca2+ channel (Ca(V)1.1). We found that Rad and Rem both potently inhibit L-type current in FDB fibers. However, intramembrane charge movement was only reduced in fibers transfected with Rad; charge movement for Rem-expressing fibers was virtually identical to charge movement observed in naive fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation or Ca(V)1.1's voltage-sensors, whereas Rad utilizes at least one mode that limits, voltage-sensor movement. Because Rad and Rem differ significantly only in their amino-termini, we constructed Rad-Rem. chimeras to probe the structural basis for the distinct specificities of Rad- and Rem-mediated inhibition. Using this approach, a chimera composed of the amino-terminus of Rem and the core/carboxyl-terminus of Rad inhibited L-type current without reducing charge movement. Conversely, a chimera having the amino-terminus of Rad fused to the core/carboxyl-terminus of. Rem inhibited L-type current with a concurrent reduction in charge movement. Thus, we have identified the amino-termini of Rad and Rem as the structural elements dictating the specific modes of inhibition of Ca(V)1.1

Potent inhibition of L-type Ca2+ currents by a Rad variant associated with congestive heart failure

Ca2+ influx via L-type voltage-gated Ca2+ channels supports the plateau phase of ventricular action potentials and is the trigger for excitation-contraction (EC) coupling in the myocardium. Rad, a member of the RGK (Rem, Rem2, Rad, Gem/Kir) family of monomeric G proteins, regulates ventricular action potential duration and EC coupling gain through its ability to inhibit cardiac L-type channel activity. In this study, we have investigated the potential dysfunction of a naturally occurring Rad variant (Q66P) that has been associated with congestive heart failure in humans. Specifically, we have tested whether Rad Q66P limits, or even eliminates, the inhibitory actions of Rad on Ca(V)1.2 and Ca(V)1.3, the two L-type channel isoforms known to be expressed in the heart. We have found that mouse Rad Q65P (the murine equivalent of human Rad Q66P) inhibits L-type currents conducted by Ca(V)1.2 or Ca(V)1.3 channels as potently as wild-type Rad (>95% inhibition of both channels). In addition, Rad Q65P attenuates the gating movement of both channels as effectively as wild-type Rad, indicating that the Q65P substitution does not differentially impair any of the three described modes of L-type channel inhibition by RGK proteins. Thus, we conclude that if Rad Q66P contributes to cardiomyopathy, it does so via a mechanism that is not related to its ability to inhibit L-type channel-dependent processes per se. However, our results do not rule out the possibility that decreased expression, mistargeting or altered regulation of Rad Q66P may reduce the RGK protein's efficacy in vivo. (C) 2013 Elsevier Inc. All rights reserved

Rad is an Agent of Skeletal Muscle Atrophy

Rad, a member of the RGK (Rad, Rem, Rem2, Gem/Kir) family of small GTP binding proteins, is expressed at very low levels in “normal” skeletal muscle. However, Rad expression is substantially upregulated in muscle of sporadic ALS patients and two established familial ALS mouse models just prior to presentation of symptoms. Rad is a potent, endogenous inhibitor of skeletal muscle L-type Ca2+ channels (CaV1.1). Recently, downregulation of CaV1.1 expression via exon skipping was reported to cause profound atrophy and fibrosis. In light of the increased Rad expression in ALS muscle, we investigated the effect(s) of long-term elevated Rad expression in otherwise normal skeletal muscle. To do so, we injected a serotype 1 Adeno-Associated Virus encoding a muscle-specific, tMCK promoter-driven Venus-Rad fusion construct into various mouse hindlimb muscles. Five months post-injection, we observed profound atrophy of V-Rad-expressing tibialis anterior (TA) muscles. These TA muscles displayed extensive fibrosis and considerable reductions in fiber diameter and area relative to naïve and GFP-expressing control TA muscles. Examination of V-Rad-expressing extensor digitorum longus fibers in thin sections revealed a general disruption of muscle ultrastructure. L-type currents and intramembrane charge movement were both reduced (∼55% and ∼34%, respectively) in V-Rad-expressing flexor digitorum brevis (FDB) fibers in comparison to naïve and GFP-expressing fibers. Likewise, depolarization-induced myoplasmic Ca2+ transients were reduced (∼55%) in V-Rad-expressing FDB fibers. The reduction in transient amplitude appeared not be a consequence of a reduced SR Ca2+ store because control and V-Rad-expressing FDB fibers had similar responses to 4-cholo-m-cresol (p > 0.05). Taken together, our results indicate that Rad can engage atrophic signaling in skeletal muscle, perhaps as an effect of inactivity due to impaired EC coupling. Supported by the Boettcher Foundation (R.A.B.), 2T32AG000279-11 (R.S. Schwartz) and 2PO1 AR052354 (P.D. Allen PI; CFA Core D)

Putative malignant hyperthermia mutation CaV1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RYR1 in skeletal muscle. Here we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant MH when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, HOM) express similar levels of CaV1.1 by qPCR, western blot, [3H]PN-200 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of TRPC3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant MH response to halothane and/or heat stress in HET and HOM mice

Calcium Channel Dysfunction in a Mutant Mouse Model of Malignant Hyperthermia(CaV1.1R174W)

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that is triggered by exposure to volatile anesthetics. MH has been studied extensively in mice and pigs carrying causative mutations in the type 1 ryanodine receptor (RyR1). However, no in vivo information exists regarding how mutations in the skeletal muscle L-type Ca2+ channel (CaV1.1) precipitate MH crises. For this reason, we generated a mouse line carrying the R174W mutation. Homozygous R174W mice ambulated efficiently, reproduced and had normal lifespans. When exposed to isoflurane, homozygous R174W mice entered a hypermetabolic state ending ultimately in death. On the ultrastructural level, R174W muscle displayed limited and variable changes: some variability of the SR calsequestrin content, displacement of mitochondria in some soleus fibers of aged mice and occasional accumulation of SR stacks. On the cellular level, homozygous R174W muscle had elevated resting myoplasmic Ca2+ levels that were greatly increased upon exposure to isoflurane. Flexor digitorum brevis (FDB) fibers dissociated from homozygous R174W mice lacked L-type Ca2+ current even though intramembrane charge movements of were of similar magnitude and voltage-dependence to those recorded from wild-type fibers. Ca2+ released from the SR in response to depolarization was substantially reduced in homozygous R174W fibers suggesting a depleted SR Ca2+ store. Lipid bilayer recordings showed that the Po of RyR1s isolated from homozygous R174W mice was significantly increased at all cis Ca2+ concentrations (Feng et al., this meeting). Taken together, our results support a mechanism for MH susceptibility in which CaV1.1 R174W promotes SR Ca2+ leak without affecting EC coupling per se. This work was supported by grants from the NIH (AR055104 to KGB, AR052534 to PDA, KGB, PMH, CFA and INP) and MDA (MDA277475 to KGB). DB received a stipend from 2T32AG000279-11

Calcium Channel Dysfunction in a Mutant Mouse Model of Malignant Hyperthermia(CaV1.1 R174W)

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that is triggered by exposure to volatile anesthetics. MH has been studied extensively in mice and pigs carrying causative mutations in the type 1 ryanodine receptor (RyR1). However, no in vivo information exists regarding how mutations in the skeletal muscle L-type Ca2+ channel (CaV1.1) precipitate MH crises. For this reason, we generated a mouse line carrying the R174W mutation. Homozygous R174W mice ambulated efficiently, reproduced and had normal lifespans. When exposed to isoflurane, homozygous R174W mice entered a hypermetabolic state ending ultimately in death. On the ultrastructural level, R174W muscle displayed limited and variable changes: some variability of the SR calsequestrin content, displacement of mitochondria in some soleus fibers of aged mice and occasional accumulation of SR stacks. On the cellular level, homozygous R174W muscle had elevated resting myoplasmic Ca2+ levels that were greatly increased upon exposure to isoflurane. Flexor digitorum brevis (FDB) fibers dissociated from homozygous R174W mice lacked L-type Ca2+ current even though intramembrane charge movements of were of similar magnitude and voltage-dependence to those recorded from wild-type fibers. Ca2+ released from the SR in response to depolarization was substantially reduced in homozygous R174W fibers suggesting a depleted SR Ca2+ store. Lipid bilayer recordings showed that the Po of RyR1s isolated from homozygous R174W mice was significantly increased at all cis Ca2+ concentrations (Feng et al., this meeting). Taken together, our results support a mechanism for MH susceptibility in which CaV1.1 R174W promotes SR Ca2+ leak without affecting EC coupling per se. This work was supported by grants from the NIH (AR055104 to KGB, AR052534 to PDA, KGB, PMH, CFA and INP) and MDA (MDA277475 to KGB). DB received a stipend from 2T32AG000279-11