title: Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel β4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.
abstract: Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory β subunits. All RGK proteins bind to voltage-gated Ca(2+) channel β subunit (Cavβ) 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 Cavβ4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the β4 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 α1A subunit (Cav2.1) Ca(2+) currents were completely inhibited by Rem2 when co-expressed with wild-type Cavβ4a, but were unaffected by Rem2 when coexpressed with a Cavβ4a site 1 (L173A/V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cavβ4 interaction and show definitively that the interaction is required for Cav2.1 inhibition.
authors.affiliation: *Department of Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA; and Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Xingfu
authors.initials: X
authors.lastname: Xu
authors.affiliation: *Department of Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA; and Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Fangxiong
authors.initials: F
authors.lastname: Zhang
authors.affiliation: *Department of Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA; and Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Zamponi
authors.affiliation: *Department of Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA; and Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada hornewil@cvm.msu.edu.
authors.forename: William A
authors.initials: WA
authors.lastname: Horne
mesh_terms: D020864:Calcium Channels, N-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods*; D004594:Electrophysiology; D057809:HEK293 Cells; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs
chemical_list: D020864:Calcium Channels, N-Type; D007473:Ion Channels; D020559:Monomeric GTP-Binding Proteins; C528471:REM2 protein, human
keywords: RGK proteins; brain; electrophysiology; ion channel; synaptic transmission