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Functional Role of a C-Terminal G-Binding Domain of Ca_v2.2 Channels

Department of Pharmacology, University of Washington, Seattle, Washington

Presynaptic Ca²⁺ channels are inhibited by neurotransmitters acting through G protein-coupled receptors via a membrane-delimited pathway. Inhibition is reversed by strong depolarization, resulting in prepulse facilitation. Activated G protein subunits (G) are required for maximal prepulse facilitation. G binds to multiple sites on Ca_v2.1, Ca_v2.2, and Ca_v2.3 1 subunits. Here we examine the functional relevance of a C-terminal binding site for G on Ca_v2.2b channels, which mediate N-type Ca²⁺ currents. In vitro binding studies showed that G subunits bind to the intracellular loop connecting domains I and II and the C-terminal domain of Ca_v2.2b but not the intracellular loops connecting domains II and III or III and IV. Deletion analysis revealed that the binding site is located near the C terminus, within amino acid residues 2257 to 2336. Directed yeast two-hybrid analysis confirmed this specific binding interaction in vivo in yeast cells. Ca_v2.2b channels with this site deleted had normal function properties, and they were inhibited essentially normally by strong activation of G proteins with guanosine 5'-3-O-(thio)triphosphate (GTPS) and were facilitated nearly normally by depolarizing prepulses. Similarly deletion of this site had small, statistically insignificant effects on inhibition of Ca²⁺ current and on prepulse facilitation in the presence of somatostatin to stimulate receptor-mediated activation of G proteins. In contrast, deletion of the C-terminal G site substantially reduced the low level of intrinsic prepulse facilitation present at the basal level of G protein activation in tsA-201 cells. Thus, this C-terminal G binding site contributes to the affinity or efficacy of G regulation at basal levels of G protein activation. The simplest interpretation of our results is that the C-terminal binding site increases the affinity of G for the channel but is not required for G action. C-terminal binding of G may influence the physiological responsiveness of Ca²⁺ channels to low-level G protein activation.

Address correspondence to: Dr. William A. Catterall, Professor and Chair, Department of Pharmacology, University of Washington, Box 357280, Seattle, WA 98195-7280. E-mail: wcatt{at}u.washington.edu

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