Determinants of the voltage dependence of G protein modulation within calcium channel β subunits

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gβγ-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVβ subunit with the CaVα1 subunit, when such interaction was prevented by a mutation in the α1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984–6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVβ subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVβ subunit that is required for this process. We have coexpressed the CaVβ subunit constructs with CaV2.2 and α2δ-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVβ subunit GK domains, from either β1b or β2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from β2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gβγ binding affinity, leading to greater tonic modulation by basal levels of Gβγ. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVβ subunit GK domains alone

Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs

Voltage-gated calcium channel �1 subunits consist of four domains (I–IV), each with six transmembrane segments. A number of truncated isoforms have been identified to occur as a result of alternative splicing or mutation. We have examined the functional consequences for expression of full-length Ca v2.2 (�1B) of its coexpression with truncated constructs of Ca v2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits �1b and �2�-1, did not form functional channels. When they were coexpressed, lowdensity whole-cell currents and functional channels with properties similar to wild-type channels were observed. However, when domain I-II, domain III-IV, or domain I alone were coexpressed with full-length Ca v2.2, they markedly suppressed its functional expression, although at the single channel level, when channels were recorded, there were no differences in thei