On the mechanism of G protein βγ subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane: Comparison with the ATP-sensitive K+ channel

Abstract

The mechanism of G protein βγ subunit (Gβγ)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. Gβγ and GTP-γS-bound α subunits (Gα*'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, Gβγ activated the KACh channel in a concentration-dependent fashion. 10 nM Gβγ almost fully activated the channel in 132 of 134 patches (98.5%). The Gβγ-induced maximal channel activity was equivalent to or sometimes larger than the GTP-γS-induced one. Half-maximal activation occurred at ∼6 nM Gβγ. Detergent (CHAPS) and boiled Gβγ preparation could not activate the KACh channel. Gβγ suspended by Lubrol PX instead of CHAPS also activated the channel. Even when Gβγ was pretreated in Mg2+-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating Giα*'s, the Gβγ activated the channel. Furthermore, Gβγ preincubated with excessive GDP-bound Goα, did not activate the channel. These results indicate that Gβγ itself, but neither the detergent CHAPS nor contaminating Giα*, activates the KACh channel. Three different kinds of Giα* 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-γS or Gβγ. Thus, Giα* activation of the KACh channel may not be significant. On the other hand, Giα*'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 μM Mg·ATP. Gβγ inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. Giα*'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. Gβγ subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: Giα activation of the KATP channel and Gβγ activation of the KACh channel.