Investigation on regulatory mechanisms for smooth muscle receptor-operated Ca^<2+> permeable cation channels using lipid bilayer incorporation of plasma membrane vesicles.

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We have established the protocol of reconstituting G-protein-coupled receptor activated cation channels into the lipid bilayer, with plasma membrane vesicles prepared from guinea-pig ileal smooth muscle using the purification technique previously applied to the large conductance Ca^<2+>-dependent and ATP-sensitive K^+ channels (Toro et al., 1990). Ultracentrifugation of crude plasma membrane vesicles across the five discontinuous sucrose gradients provided a plasma membrane fraction (at 25/30% (w/w) sucrose interface) highly enriched with plasmalemmal proteins. Under Na^+-rich conditions, incorporation of these plasma membrane vesicles into the bilayer produced GTPγS (100μM)-activatable channel activities that are inhibited by GDPβS (1mM), sensitive to Ca^<2+> and enhanced by depolarization. The reversal potential and unitary conductance (tens of picosiemens) of these channels varied depending on Na^+ concentration, but not affected by Cl^-. These results strongly indicate that the reconstituted channels activated by GTPγS belong to a class of voltage-dependent, Ca^<2+>-sensitive cation-selective channels that are activated through a G-protein, and correspond most likely to the muscarinic receptor-activated cation channels previously identified in the same preparation. These results also strongly point to the usefulness of bilayer incorporation technique to investigate the receptor-operated cation channels in smooth muscle in the future.