Christin F. Romberg

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.

Apparent lack of physical or functional interaction between CaV1.1 and its distal C terminus

The distal C-terminal domain of CaV1.1 is not required for depolarization-induced potentiation of L-type Ca2+ current in skeletal muscle.

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.