RGK Proteins Inhibit slow, Depolarization-Dependent CA2+ Entry into Cultured Myotubes

Abstract

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (CaV1.1): 1) voltage-sensor for excitation-contraction (EC) coupling, 2) L-type Ca2+ channel, and 3) voltage-sensor for slow, depolarization-dependent Ca2+ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of CaV1.1. However, it is not known whether the latter function that has been attributed to CaV1.1 is subject to modulation by RGK proteins. The purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly activating, persistent Ca2+ entry that is dependent on the voltage-sensing capability of CaV1.1. To investigate this possibility, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in otherwise normal mouse myotubes and the abilities of each of these V-RGK proteins to inhibit EC coupling, L-type Ca2+ current and depolarization-dependent Ca2+ entry in myotubes were assessed using electrical field stimulation, whole-cell voltage-clamp and Ca2+ imaging, respectively. As shown previously for YFP-Rem, both EC coupling and L-type current density were substantially attenuated in developing myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca2+ entry (89%, 99% and 91% for V-Rad, V-Rem and V-Gem, respectively, relative to control). Thus, we provide the first evidence of modulation of this enigmatic type of Ca2+ entry peculiar to skeletal muscle. Moreover, the similar inhibitory effects of RGK proteins on both L-type current amplitude and slow Ca2+ entry provide further evidence that both modes of Ca2+ flux utilize a common pathway. Supported by AG038778 (to RAB).