G-PROTEIN-DEPENDENT FACILITATION OF NEURONAL ALPHA(1A), ALPHA(1B) ANDALPHA(1E) CA CHANNELS

Modulation of neuronal voltage-gated Ca channels has important implica tions for synaptic function. To investigate the mechanisms of Ca chann el modulation, we compared the G-protein-dependent facilitation of thr ee neuronal Ca channels. alpha(1A), beta(1B) or alpha(1E) subunits wer e transiently coexpressed with alpha(2)-delta(b) and beta(3) subunits in HEK293 cells, and whole-cell currents were recorded. After intracel lular dialysis with GTP gamma S, strongly depolarized conditioning pul ses facilitated currents mediated by each Ca channel type. The magnitu de of facilitation depended on current density, with low-density curre nts being most strongly facilitated and high-density currents often la cking facilitation. Facilitating depolarizations speeded channel activ ation similar to 1.7-fold for alpha(1A) and alpha(1B) and increased cu rrent amplitudes by the same proportion, demonstrating equivalent faci litation of G-protein-inhibited alpha(1A) and alpha(1B) channels. Inac tivation typically obscured facilitation of alpha(1E) current amplitud es, but the activation kinetics of alpha(1E) currents showed consisten t and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha(1A), alpha(1B) and al pha(1E) suggesting that G beta gamma dimers dissociate from and reasso ciate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mut ant of alpha(1B) missing large segments of the II-III loop and C termi nus. This deletion mutant exhibited undiminished G-protein-dependent f acilitation, demonstrating that a G beta gamma interaction site recent ly identified within the C terminus of alpha(1E) is not required for m odulation of alpha(1B).