Reconstitution of Somatostatin and Muscarinic Receptor Mediated Stimulation of K⁺Channels by Isolated G_KProtein 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 pipette to 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 a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)

The human genome encodes at least three non‐allellic G proteins with αi‐type subunits

“…[3,4,7,8]) were also called Gi. It is now known, however, that PTX affects not only inhibition of adenylyl cyclase but, depending on the system studied, also receptorand GTP-dependent activation of phospholipases of the C and/or AZ type [20-241 and activation of K+ channels [25,26]. Since inhibition of adenylyl cyclase and activation of phospholipases are regulated by receptors independently of each other, this suggests that more than one PTXsensitive G protein should exist.…”

“…ai-subunits of M, 41000 are encoded in type-l ai cDNA [5,6] and differ in at least 23 of its 354 amino acids from type-3 ai reported here and in at least 45 amino acids from type-2 ais. The human erythrocyte 'putative Ni' (a-subunit of M, 40000 [2,33]) is active in stimulating the opening of K+ channels [25,26] through its m-subunit [34,35] and has been re-named by us Gk. Gi proteins with ais of M, -40000…”

G(o)‐2 protein mediates the reduction in Ca2+ currents by somatostatin in cultured ovine somatotrophs.

“…The effect of SRIF on voltage-gated Ca2+ currents in rat somatotrophs is prevented by pertussis toxin pretreatment (Chen et al 1990a). G proteins also mediate the effects of SRIF on K+ channels (Yatani et al 1987) and GH secretion (Chen et al 1990a). The ai subunit of Gi protein also mediates the effect of SRIF on cAMP production in neurones (Yasuda et al 1992).…”

“…It has been suggested that G proteins mediate this response on the basis of blockade of the response by pertussis toxin treatment (Ikeda & Schofield, 1989;Surprenant et al 1990;Chen et al 1990a) and because the SRIF receptor has a structure that typically couples to G proteins . Subtypes of the receptor are thought to be coupled to different types of G protein and the Gi protein is thought to mediate the effect of SRIF on K+ channels (Yatani, Codina, Sekura, Birnbaumer & Brown, 1987 …”

Distribution and localization of a G protein‐coupled inwardly rectifying K+ channel in the rat

“…In GH, and AtT20 clonal rat anterior pituitary cells ACh and somatostatin activate a similar inwardly rectifying conductance, however with a slightly larger 55 pS conductance [18]. An 80 pS channel represents an effector for the action of opiates and possibly norepinephrine and somatostatin in the locus coeruleus [19]; multiple conductance states can be activated by serotonin in the dorsal raphe [20].…”