Reconstitution in Lipid Bilayer of Smooth Muscle Cation Channels Activated Through a GTP-Binding Protein

Abstract

Reconstitution of G-protein-coupled receptor activated cation channels into the lipid bilayer was attempted with plasma membrane vesicles prepared from guinea-pig ileal smooth muscle using the purification technique previously applied to the large conductance Ca²⁺-dependent and ATP-sensitive K⁺ channels (Toro et al., 1990). Under Na⁺-rich conditions, incorporation of plasma membrane vesicles into the bilayer produced GTPγS (100 μM)-activatable channel activities that are inhibited by GDPβS (1 mM), sensitive to Ca²⁺ and enhanced by depolarization. The reversal potential and unitary conductance (tens of picosiemens) of these channels varied in a manner dependent 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, Ca²⁺-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 suggest potential usefulness of bilayer incorporation technique to investigate the receptor-operated cation channels in smooth muscle.