Direct interaction of G   with a C-terminal G  -binding domain of the Ca2+ channel  1 subunit is responsible for channel inhibition by G protein-coupled receptors

The beta-subunit is an integral component of purified voltage-sensitive Ca2+ channels. Modulation of Ca2+ channel activity by the beta-subunit, which includes significant increases in transmembrane current and/or changes in kinetics, is observed on coexpression of six alpha 1-subunit genes with four beta-subunit genes in all alpha 1-beta combinations tested. Recent reports suggest that this regulation is not due to targeting of the alpha 1-subunit to the plasma membrane but is probably a result of a conformational change induced by the beta-subunit. Here we report that the beta-subunit binds to the cytoplasmic linker between repeats I and II of the dihydropyridine-sensitive alpha 1-subunits from skeletal (alpha 1S) and cardiac muscles (alpha 1C-a), and also with the more distantly related neuronal alpha 1A and omega-conotoxin GVIA-sensitive alpha 1B-subunits. Sequence analysis of the beta-subunit binding site identifies a conserved motif (QQ-E--L-GY--WI--E) positioned 24 amino acids from the IS6 transmembrane domain in each alpha 1-subunit. Mutations within this motif reduce the stimulation of peak currents by the beta-subunit and alter inactivation kinetics and voltage-dependence of activation. Conservation of the beta-subunit binding motif in these functionally distinct calcium channels suggests a critical role for the I-II cytoplasmic linker of the alpha 1-subunit in channel modulation by the beta-subunit.

The most commonly used signal transduction pathway for receptor-mediated N-type Ca2+-channel modulation involves activation of a heterotrimeric G protein to produce voltage-dependent inhibition. Although it is widely assumed that Galpha mediates this effect, experiments to address this hypothesis directly are lacking. Here I show that transient overexpression of Gbetagamma in sympathetic neurons mimics and occludes the voltage-dependent Ca2+ channel modulation produced by noradrenaline (NA). Conversely, over-expression of Galpha produces minimal effects on basal Ca2+ channel behaviour but attenuates NA-mediated inhibition in a manner consistent with the buffering of Gbetagamma. These observations indicate that it is Gbetagamma, and Galpha, that mediates voltage-dependent inhibition of N-type Ca2+ channels. The identification of Gbetagamma as the mediator of this pathway has broad implications as G-protein-coupled receptors, many of which are implicated in disease or are targets of therapeutic agents, couple to N-type Ca2+ channels and may modulate synaptic transmission by this mechanism.

Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation.