Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes.
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)
Yatani
A
A
Department of Physiology, Baylor College of Medicine, Houston, Texas 77030.
Codina
J
J
Sekura
R D
RD
Birnbaumer
L
L
Brown
A M
AM
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D002999:Clone Cells; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D010903:Pituitary Gland, Anterior; D010911:Pituitary Neoplasms; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D011976:Receptors, Muscarinic / Q000502:physiology*; D013004:Somatostatin / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology
D007473:Ion Channels; D011976:Receptors, Muscarinic; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin; D006160:Guanosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins; D011188:Potassium