Functional assessment of three Rem residues identified as critical for interactions with Ca2+ 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 alpha(1) and the auxiliary beta 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 beta subunits. However, the functional significance of these residues is not known. To investigate the contributions of R200, L227, and H229 to beta 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 Ca(V)2.2 alpha(1B), beta(3), and alpha(2)delta-1 channel subunits. The V-Rem L227A single mutant also failed to reduce N-type currents conducted by coexpressed Ca(V)2.2 channels, a finding consistent with the previous observation that a leucine at position 227 is critical for Rem-beta interactions. Rem-dependent inhibition of Ca(V)2.2 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 beta(3) subunits in vitro, V-Rem H229A produced nearly complete inhibition of Ca(V)2.2-mediated currents.