title: Functional assessment of three Rem residues identified as critical for interactions with Ca(2+) channel β subunits.
abstract: Members of the Rem, Rem2, Rad, Gem/Kir (RGK) family of small GTP-binding proteins inhibit high-voltage-activated (HVA) Ca(2+) 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 Ca(2+) 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.
authors.affiliation: Department of Medicine-Cardiology Division, School of Medicine, University of Colorado Denver-Anschutz Medical Campus, 12700 East 19th Avenue, P15-8006, B-139, Aurora, CO, 80045, USA.
authors.forename: Donald
authors.initials: D
authors.lastname: Beqollari
authors.affiliation: Department of Medicine-Cardiology Division, School of Medicine, University of Colorado Denver-Anschutz Medical Campus, 12700 East 19th Avenue, P15-8006, B-139, Aurora, CO, 80045, USA.
authors.forename: Christin F
authors.initials: CF
authors.lastname: Romberg
authors.affiliation: Department of Vegetative Physiology, University of Cologne, Robert-Koch-Str. 39, D-50931, Cologne, Germany.
authors.forename: Dilyana
authors.initials: D
authors.lastname: Filipova
authors.affiliation: Department of Vegetative Physiology, University of Cologne, Robert-Koch-Str. 39, D-50931, Cologne, Germany.
authors.forename: Symeon
authors.initials: S
authors.lastname: Papadopoulos
authors.affiliation: Department of Medicine-Cardiology Division, School of Medicine, University of Colorado Denver-Anschutz Medical Campus, 12700 East 19th Avenue, P15-8006, B-139, Aurora, CO, 80045, USA. roger.bannister@ucdenver.edu.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Bannister
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011817:Rabbits; D051381:Rats
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D020559:Monomeric GTP-Binding Proteins; D000409:Alanine
keywords: CaV2.2; N-type; RGK protein; Rem; α1B; β subunit