Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway intrinsic to the membrane. Here we show that this inhibition results from a direct interaction between the G-protein betagamma complex and the pore-forming alpha1 subunits of several types of these channels. The interaction is mediated by the cytoplasmic linker connecting the first and second transmembrane repeats. Within this linker, binding occurs both in the alpha1 interaction domain (AID), which also mediates the interaction between the alpha1 and beta subunits of the channel, and in a second downstream sequence. Further analysis of the binding site showed that several amino-terminal residues in the AID are critical for Gbetagamma binding, defining a site distinct from the carboxy-terminal residues shown to be essential for binding the beta-subunit of the Ca2+ channel. Mutation of an arginine residue within the N-terminal motif abolished betagamma binding and rendered the channel refractory to G-protein modulation when expressed in Xenopus oocytes, showing that the interaction is indeed responsible for G-protein-dependent modulation of Ca2+ channel activity.
1. Voltage-dependent Ca2+ channels are multi-protein complexes composed of at least three subunits: a,, a28 and f. Ba2+ currents were recorded in Xenopus oocytes expressing the neuronal a1A Ca2+ channel, using the two-electrode voltage-clamp technique. Various subunit combinations were studied: a1A a1Aa2l&b, cxlAf or '11Aa-2b,ft.2. The a1A subunit alone directs the expression of functional Ca2+ channels. It carries all the properties of the channel: gating, permeability, voltage dependence of activation and inactivation, and pharmacology. The alA channel is activated by low voltages when physiological concentrations of the permeant cation are used. Both ancillary subunits a28 and f induced considerable changes in the biophysical properties of the alA current. The subunit specificity of the changes in current properties was analysed for all four ft gene products by coexpressing /lbl f2a. ft3 and f4.3. All ft subunits induce a stimulation in the current amplitude, a change in inactivation kinetics, and two hyperpolarizing shifts -one in the voltage dependence of activation and a second in the voltage dependence of steady-state inactivation. The most significant difference in regulation among ft subunits is the induction of variable rate constants of current inactivation. Rates of inactivation were induced in the following order (fastest to slowest): f3 > lb = ft4 > At2a. 4. The a2 b subunit does not modify the properties of alA Ca2+ channels in the absence of ft subunits. However, this subunit increases the f-induced stimulation in current amplitude and also regulates the ft-induced change in inactivation kinetics. 5. Of all the subunit combinations tested, Ca2P channels that included a f8 subunit were the most prone to decrease in activity. It is concluded that ft subunits are the primary target for the inhibitory mechanisms involved in Ca2+ channel run-down.6. Both Z28b and flb subunits slightly modified the sensitivity of the alA subunit to the snail peptide w-conotoxin MVIIC.7. The subunit-induced changes in properties of the alA channel are surprisingly similar to changes reported for other a, subunits. These modifications in channel activity should therefore represent important functional landmarks in the on-going characterization of subunit-subunit interactions.Voltage-dependent Ca2P channels are well characterized on contains the binding sites for all Ca2+ channel modulators the basis of their biophysical and pharmacological affecting this channel type; the a28 subunit, a disulphideproperties. At least three types of high-voltage-activated linked dimer; the transmembrane y subunit and the intraCa2+ channels, termed L-, N-and P-type, have been cellular ft subunit (Takahashi, Seagar, Jones, Reber & differentiated by their sensitivities to various neurotoxins Catterall, 1987). The primary structures of all the subunits and organic Ca2+ channel modulators (Miller, 1992). The composing this L-type Ca2+ channel were deduced by purified rabbit skeletal muscle L-type channel is composed cloning (Tanabe et al. 1987...
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