G-protein-dependent facilitation of neuronal α(1A), α(1B), and α(1E) Ca channels
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abstract
Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. α(1A), α(1B), or α(1E) subunits were transiently coexpressed with α2-δ(b) and β3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPγS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation ~1.7-fold for α(1A) and α(1B) and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited α(1A) and α(1B) channels. Inactivation typically obscured facilitation of α(1E) current amplitudes, but the activation kinetics of ate currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for α(1A), α(1B), and α(1E), suggesting that Gβγ, dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of α(1B) missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G- protein-dependent facilitation, demonstrating that a Gβγ, interaction site recently identified within the C terminus of α(1E) is not required for modulation of α(1B).
