Donald Beqollari

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 GTP-binding proteins profoundly inhibit L-type Ca(2+) channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of Po 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 Ca(2+) channel (CaV1.1). 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 naïve fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation of CaV1.1s 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 CaV1.1.

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

Ca(2+) influx via L-type voltage-gated Ca(2+) 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 CaV1.2 and CaV1.3, 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 CaV1.2 or CaV1.3 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 proteins efficacy in vivo.

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

The RGK protein Rem uncouples the voltage sensors of CaV1.1 from RYR1-mediated sarcoplasmic reticulum Ca2+ release via its ability to interact with the auxiliary β1a subunit.

Functional assessment of three Rem residues identified as critical for interactions with Ca(2+) channel β 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 α1 and the auxiliary β 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 β subunits. However, the functional significance of these residues is not known. To investigate the contributions of R200, L227, and H229 to β 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 CaV2.2 α1B, β3, and α2δ-1 channel subunits. The V-Rem L227A single mutant also failed to reduce N-type currents conducted by coexpressed CaV2.2 channels, a finding consistent with the previous observation that a leucine at position 227 is critical for Rem-β interactions. Rem-dependent inhibition of CaV2.2 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 β3 subunits in vitro, V-Rem H229A produced nearly complete inhibition of CaV2.2-mediated currents.

Molecular mechanisms and physiological relevance of RGK proteins in the heart

The primary route of Ca2+ entry into cardiac myocytes is via 1,4‐dihydropyridine‐sensitive, voltage‐gated L‐type Ca2+ channels. Ca2+ influx through these channels influences duration of action potential and engages excitation‐contraction (EC) coupling in both the atria and the myocardium. Members of the RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTP‐binding proteins are potent, endogenously expressed inhibitors of cardiac L‐type channels. Although much work has focused on the molecular mechanisms by which RGK proteins inhibit the CaV1.2 and CaV1.3 L‐type channel isoforms that expressed in the heart, their impact on greater cardiac function is only beginning to come into focus. In this review, we summarize recent findings regarding the influence of RGK proteins on normal cardiac physiology and the pathological consequences of aberrant RGK activity.

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p > 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Defining the MO's of RGK proteins.

Members of the RGK (Rad-Rem-Rem2-Gem/Kir) family of small GTP binding proteins profoundly inhibit high voltage-activated CaV1.X and CaV2.X Ca2C channels. The ability of RGK proteins to interact with these channels was revealed when Gem was fished out of a yeast-2-hybrid screen which used the channel b subunit as bait. The functional significance of this interaction was immediately clear as Ca2C currents were more-or-less absent in cells coexpressing Gem with either CaV1.2, CaV1.3 or CaV2.1. Since the ablation of Ca2C currents by Gem, and later Rad, Rem and Rem2, required coexpression of a b subunit isoform, two reasonable assumptions arose regarding the nature of RGK protein-mediated inhibition. The first assumption was that RGK proteins blocked channel membrane expression by interfering with the b subunit’s well-known ability to promote channel trafficking. The second assumption was that RGK proteins interacted only with the b subunit. The first indication that first assumption might be a bit na€ıve became apparent when Chen et al. reported that HEK 293 coexpressing CaV2.2 channels and Rem2 displayed virtually no difference in maximal v-conotoxin GVIA binding relative to cells expressing just the channel. However, other evidence, such as the reduction of intramembrane gating charge movement in cardiomyocytes by virally-overexpressed Gem, still supported the idea that RGK proteins reduce channel membrane expression. These conflicting viewpoints were reconciled by Yang and colleagues who, using HEK293 cells coexpressing CaV1.2, b2a and Rem, found that Rem can support three distinct modes of inhibition: 1) reduction of channel membrane expression, 2) immobilization of the voltage-sensors, and 3) reduction of channel Po without impaired voltage-sensor movement (see Figure 1). Two relatively recent studies have challenged the second assumption that RGK proteins exert their influence on channel activity solely via interactions with the b subunit. Fan et al. washed away b subunits from insideout membrane patches to demonstrate that application of purified Gem can directly inhibit CaV2.1 channels, 5