Reconstitution of Somatostatin and Muscarinic Receptor Mediated Stimulation of K+Channels by Isolated GKProtein in Clonal Rat Anterior Pituitary Cell Membranes *

Abstract

Somatostatin (SS) inhibits secretion from many cells, including clonal GH₃ pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K⁺ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca²⁺ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipetteto the extracellular face of a patch activate a 55-picosiemens K⁺ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD⁺, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K⁺ channel can also be activated in aligand-independent manner with guanosine [γ-thio] triphosphate (GTPγS) or with Mg²⁺/GTPγS-activated erythrocyte G protein. We call this protein G_k. It is an α-β-γ trimer of which we have previously shown that the α-subunit is the substrate for PTX and that it dissociates on activation with Mg²⁺/GTPγS into α-GTPγS plus β-γ. A similarly activated and dissociated preparation of G_s, the stimulatory regulatory component of adenylyl cyclase, having a different α-subunit but the same β-γ-dimer, was unable to cause K⁺ opening. These experiments establish that a K⁺ channel-mediated membrane hyperpolarization is a primary response of a secretory cell to an inhibitory hormone, that a PTX-sensitive G protein directly couples hormone receptors to K⁺ channels in endocrine cells, and that the effect of G_k on the effector is mediated by its α-subunit.