Functional role of a C-terminal Gbetagamma-binding domain of Ca(v)2.2 channels

Abstract

Presynaptic Ca(2+) 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 betagamma subunits (Gbetagamma) are required for maximal prepulse facilitation. Gbetagamma binds to multiple sites on Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 alpha1 subunits. Here we examine the functional relevance of a C-terminal binding site for Gbetagamma on Ca(v)2.2b channels, which mediate N-type Ca(2+) currents. In vitro binding studies showed that Gbetagamma subunits bind to the intracellular loop connecting domains I and II and the C-terminal domain of Ca(v)2.2b 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(v)2.2b 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 (GTPgammaS) and were facilitated nearly normally by depolarizing prepulses. Similarly deletion of this site had small, statistically insignificant effects on inhibition of Ca(2+) 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 Gbetagamma 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 Gbetagamma binding site contributes to the affinity or efficacy of Gbetagamma 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 Gbetagamma for the channel but is not required for Gbetagamma action. C-terminal binding of Gbetagamma may influence the physiological responsiveness of Ca(2+) channels to low-level G protein activation.