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