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nmrlearner 01-08-2015 01:29 PM

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel ?4 subunit: A low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.
 
Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel ?4 subunit: A low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel ?4 subunit: A low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

FASEB J. 2015 Jan 6;

Authors: Xu X, Zhang F, Zamponi GW, Horne WA

Abstract
Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory ? subunits. All RGK proteins bind to voltage-gated Ca(2+) channel ? subunit (Cav?) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cav?4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the ?4 guanylate kinase domain contributing to a 156 ± 18 µM Kd interaction: a hydrophobic pocket lined by 4 critical residues (L173, N261, H262, and V303), mutation of any of which completely disrupted binding, and a nearby surface containing 3 residues (D206, L209, and D258) that when individually mutated decreased affinity. Voltage-gated Ca(2+) channel ?1A subunit (Cav2.1) Ca(2+) currents were completely inhibited by Rem2 when co-expressed with wild-type Cav?4a, but were unaffected by Rem2 when co-expressed with a Cav?4a site 1 (L173A/V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cav?4 interaction and show definitively that the interaction is required for Cav2.1 inhibition.-Xu, X., Zhang, F., Zamponi, G. W., Horne, W. A. Solution NMR and calorimetric analysis of Rem2 binding to the Ca(2+) channel ?4 subunit: A low affinity interaction is required for inhibition of Cav2.1 Ca(2+) currents.


PMID: 25563298 [PubMed - as supplied by publisher]



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