Mechanism for reactivation of the ATP-sensitive K+ channel by MgATP complexes in guinea-pig ventricular myocytes.

Abstract

1. A mechanism underlying reactivation of the adenosine 5'-triphosphate-sensitive K+ (K+ATP) channels by MgATP complexes after run-down was examined in guinea-pig ventricular myocytes using the patch-clamp technique with inside-out patch configuration. 2. After run-down was induced by exposure of the intracellular side of the membrane patch to Ca2+ (1 mM), channel activity was reactivated by exposure and subsequent wash-out of MgATP (2 mM). Addition of inhibitors of various serine/threonine protein kinases to the MgATP solution did not suppress reactivation of the run-down channels. 3. Non- or poorly hydrolysable ATP analogues were unable to reactivate run-down channels. 4. The degree of channel recovery was dependent upon the duration of MgATP exposure. The apparent half-activation value (K1/2) of MgATP for reactivation was decreased with increasing exposure time. 5. Various products of ATP hydrolysis were unable to reactivate run-down channels except a relatively low concentration (100 microM) of ADP exposure. 6. Other nucleotide triphosphates, in the presence of Mg2+, were unable to reactivate rundown channels. 7. Fluorescein 5-isothiocyanate (50 microM), which interacts with lysine residues of the nucleotide-binding site on various ATPases, inhibited K+ATP channel activity. After wash-out, channel activity recovered only slightly. 8. These data suggest that the hydrolysis of ATP is important for reactivation of run-down K+ATP channels but that protein phosphorylation by serine/threonine protein kinases may not be involved. Since no products of ATP hydrolysis could reproduce MgATP-induced channel reactivation and since the degree of channel recovery was dependent upon the duration of MgATP application, the hydrolysis energy appears to be utilized for channel reactivation.