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A Low Affinity Cavb4a/Rem2 Interaction is Required for Inhibition of Cav2.1 Ca2+ Currents
Author(s) -
Xu Xingfu,
Zhang Fangxiong,
Zamponi Gerald,
Horne William
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.714.13
Subject(s) - mutant , intracellular , binding site , chemistry , biophysics , in vitro , mutation , plasma protein binding , microbiology and biotechnology , biochemistry , biology , gene
RGK (Rem, Rad, Kir/Gem proteins) proteins, including Rem2, cause profound inhibition of high‐voltage activated Ca 2+ channels containing intracellular regulatory b subunits. All RGK proteins bind to Cavb subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies solution state nuclear magnetic resonance (NMR) and calorimetric techniques to map the binding site for Rem2 on human Cavb4a and to measure the binding affinity. Our experiments revealed two binding surfaces on the b4 GK domain contributing to a 156 μM Kd interaction: 1) a hydrophobic pocket surrounded by four residues, mutation of any of which completely disrupted binding; and 2) a nearby surface containing three residues that when individually mutated decreased affinity. Cav2.1 Ca 2+ currents were completely inhibited by Rem2 when co‐expressed with wild‐type Cavb4a, but were unaffected by Rem2 when co‐expressed with a Cavb4a site 1 mutant. These results provide direct evidence for a low affinity Rem2/Cavb4 interaction and its essential role in Cav2.1 inhibition by Rem2.