Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3–5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca2+ channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation. Methods: We recorded L-type currents, charge movements, and myoplasmic Ca2+ transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1G93A). Results: In FDB fibers obtained from “symptomatic” global SOD1G93A mice, we observed a substantial reduction of SR Ca2+ release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1G93A fibers when compared to control fibers. Ca2+ transients were not significantly reduced in similar experiments performed with FDB fibers obtained from “early-symptomatic” SOD1G93A mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca2+ release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1G93A expression was restricted to skeletal muscle. Conclusions: We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1G93A mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1G93A and skeletal muscle autonomous MLC/SOD1G93A mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status

Alternative RNA splicing: contribution to pain and potential therapeutic strategy

Since the sequencing of metazoan genomes began, it has become clear that the number of expressed proteins far exceeds the number of genes. It is now estimated that greater up to 98% of human genes give rise to multiple proteins through alternative pre-mRNA splicing. This review highlights the known alternative splice variants of many channels, receptors and growth factors important in nociception and pain. Recently, pharmacological control of alternative splicing has been proposed as potential therapy in cancer, wet age-related macular degeneration, retroviral infections and pain. In this review we consider the effects that known splice variants of molecules key to nociception/pain have on nociceptive processing and/or analgesic action, and the potential for control of alternative pre-mRNA splicing as a novel analgesic strategy

Functional assessment of three Rem residues identified as critical for interactions with Ca2+ channel beta subunits

Members of the Rem, Rem2, Rad, Gem/Kir (RGK) family of small GTP-binding proteins inhibit high-voltage-activated (HVA) Ca2+ channels through interactions with both the principal alpha(1) and the auxiliary beta subunits of the channel complex. Three highly conserved residues of Rem (R200, L227, and H229) have been shown in vitro to be critical for interactions with beta subunits. However, the functional significance of these residues is not known. To investigate the contributions of R200, L227, and H229 to beta subunit-mediated RGK protein-dependent inhibition of HVA channels, we introduced alanine substitutions into all three positions of Venus fluorescent protein-tagged Rem (V-Rem AAA) and made three other V-Rem constructs with an alanine introduced at only one position (V-Rem R200A, V-Rem L227A, and V-Rem H229A). Confocal imaging and immunoblotting demonstrated that each Venus-Rem mutant construct had comparable expression levels to Venus-wild-type Rem when heterologously expressed in tsA201 cells. In electrophysiological experiments, V-Rem AAA failed to inhibit N-type Ca2+ currents in tsA201 cells coexpressing Ca(V)2.2 alpha(1B), beta(3), and alpha(2)delta-1 channel subunits. The V-Rem L227A single mutant also failed to reduce N-type currents conducted by coexpressed Ca(V)2.2 channels, a finding consistent with the previous observation that a leucine at position 227 is critical for Rem-beta interactions. Rem-dependent inhibition of Ca(V)2.2 channels was impaired to a much lesser extent by the R200A substitution. In contrast to the earlier work demonstrating that Rem H229A was unable to interact with beta(3) subunits in vitro, V-Rem H229A produced nearly complete inhibition of Ca(V)2.2-mediated currents

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