A B ST R AC T The mechanism of G protein ~3,/subunit (Gl3v)-induced activation of the muscarinic K + channel (KAch) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G0v and GTP-~,S-bound ct subunits (G*ds) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg 2+, GI~ ~ activated the KACh channel in a concentration-dependent fashion. 10 nM G~ almost fully activated the channel in 132 of 134 patches (98.5%). The Gl~-induced maximal channel activity was equivalent to or sometimes larger than the GTP-TS-induced one. Half-maximal activation occurred at ~ 6 nM Gt3 ~. Detergent (CHAPS) and boiled G~ preparation could not activate the KACh channel. G~v suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G~ was pretreated in Mg2+-free EDTA internal solution containing GDP analogues (24---48 h) to inactivate possibly contam0t' inating G~ s, the G0v activated the channel. Furthermore, GI~ ~ preincubated with excessive GDP-bound Go,, did not activate the channel. These results indicate that GI~ v itself, but neither the detergent CHAPS nor contaminating G~, activates the KACh channel. Three different kinds of G,*, at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was ~ 20% of that induced by GTP-~/S or Gay.

SUMMARY1. Effects of intracellular nucleotide diphosphates (NDPs) on the ATP-sensitive K+ channel (KkTP channel) were examined in ventricular cells of guinea-pig heart, using the inside-out patch clamp technique. On formation of inside-out patches in the ATP-free internal solution, the K+TP channel appeared and then ran down spontaneously. This run-down of the KATP channel activity was probably due to dephosphorylation.2. Millimolar concentrations of various NDPs, e.g. UDP (uridine diphosphate), IDP (inosine diphosphate), CDP (cytidine diphosphate) and GDP (guanosine diphosphate), applied to the internal side of the patch membrane, induced openings of the KkTP channel after run-down, i.e. in the dephosphorylated state. ADP opened the channel weakly at low concentrations (100 gM) but inhibited it at higher concentrations (1-10 mM).

No abstract

No abstract