RGK proteins: fashioning muscle with “Rad” new brakes.

Members of the Ras superfamily of monomeric GTP-binding proteins (RGK proteins: Rad, Rem, Rem2, Gem/Kir) are potent inhibitors of multimeric, high voltage-activated Ca2+ channels (CaV1 and CaV2). Specifically, RGK proteins inhibit voltage-gated Ca2+ currents mediated by cardiac CaV1.2, neuroendocrine/sinoatrial node CaV1.3, and neuronal CaV2.1 and CaV2.2 channels. However, the precise degree and mechanism of inhibition depends on the particular RGK protein, CaV channel, and cell type involved, consistent with distinct roles of RGK proteins in modulating Ca2+ signaling and excitability in different tissues.

RGK proteins inhibit CaV channels via at least three distinct molecular mechanisms including reducing channel: 1) surface expression, 2) open probability independent of voltage sensor (“charge”) movement, and 3) open probability due to partial charge immobilization. This complexity of inhibition is enhanced by the fact that RGK proteins bind to both CaV channel α1- and β-subunits and that only mechanism 3 requires GTP binding to the RGK protein. RGK proteins do not inhibit low voltage-activated Ca2+ currents mediated by CaV3 channels, in which channel assembly, trafficking, and gating does not require auxiliary subunits. However, while the interaction of RGK proteins with CaV channel β-subunits represents a key aspect of channel inhibition, β-subunit-independent components of RGK-mediated CaV1.2 channel inhibition have been demonstrated.

The importance of RGK proteins in skeletal muscle has long been appreciated. Rad was first identified as a protein increased in muscle of individuals with type II diabetes and Rem overexpression inhibits depolarization-induced Ca2+ release in C2C12 myotubes. CaV1.1 in skeletal muscle serves the following three important functions: 1) a voltage sensor for excitation-contraction (EC) coupling (voltage sensor function), 2) a voltage-gated, L-type Ca2+ channel activity (channel function), 3) a protein required for a slow form of depolarization-induced Ca2+ influx termed excitation-coupled Ca2+ entry (ECCE function). Rem inhibits CaV1.1 currents, charge movements, and voltage-gated Ca release following transient expression in primary mouse myotubes, consistent with inhibition of both CaV1.1 voltage sensor and channel functions. In this issue of Channels, Romberg and co-authors demonstrate that Ca2+ currents in myotubes are also strongly inhibited by Rad and Gem RGK proteins and that all three RGK proteins abolish ECCE. The parallel inhibition of L-type Ca2+ currents and ECCE by three different RGK proteins provides strong evidence that Ca2+ entry through CaV1.1 underlies ECCE.

The precise molecular mechanism(s) by which RGK proteins inhibit the three functions of CaV1.1 in skeletal muscle remains unclear. Rem inhibits Ca2+ currents and charge movements in myotubes. In adult muscle, however, Rem reduces Ca2+ currents in the absence of an effect on charge movement, while Rad reduces both Ca2+ currents and charge movement. The difference in the effect of Rem and Rad on CaV1.1 charge movement in adult muscle is conferred by their respective RGK N-termini. Thus, RGK protein inhibition of CaV1.1 channels depends on the specific RGK protein and cellular context involved. It is unclear if RGK inhibition of CaV1.1 channel function involves direct interactions of RGK proteins with the α1-subunit, the β-subunit, or interactions with both subunits (Fig. 1). RGK proteins could also interact with other key regulatory proteins within the triad junction. For example, stac3, a newly identified protein essential skeletal muscle EC coupling, binds both the α1-subunit of CaV1.1 and the ryanodine receptor Ca2+ release channel. Thus, future studies designed to determine interactions between specific RGK proteins with the different components of the muscle EC coupling machinery will provide important new insights into the molecular mechanisms by which RGK proteins inhibit CaV1.1 function as a voltage sensor, Ca2+ channel, and mediator of ECCE.

Figure 1. Potential interactions of RGK proteins with the CaV1.1 channel complex. RGK proteins inhibit CaV1.1 Ca2+ currents (green lines), charge movements (dashed black lines), and voltage-gated Ca2+ release (purple lines). Dashed lines are used to represents inhibitory CaV1.1 pathways shown to be mediated by some, but not, all RGK proteins. CaV1.1 inhibition may occur through RGK interactions with either the α1S- and/or β1a−subunits of the CaV1.1 channel complex, though specific sites of interaction are unknown (putative sites of interaction are indicated using question marks). ECC, excitation-contraction coupling; T-tubule, transverse tubule; RyR1, type I ryanodine receptor; SR, sarcoplasmic reticulum.

Romberg et al. show that RGK tripartite inhibition of CaV1.1 function leads to altered myotube morphology and contractile performance. While RGK expression is low in healthy muscle, Rad expression in muscle is increased in some forms of human disease, such as type II diabetes. In addition, CaV1.1-dependent charge movement and voltage-gated Ca2+ release are reduced in aged skeletal muscle, raising the possibility that reduced muscle performance in aging could also involve a component due to the increased RGK expression in muscle. Future work is needed to determine if this putative “RGK-mediated CaV1.1 brake” contributes to muscle dysfunction in aging and disease or if this mechanism serves a protective function to limit Ca2+ overload and muscle fiber degeneration.