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+ (KATP) 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 (K%) 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 /M) of ADP exposure. 6. Other nucleotide triphosphates, in the presence of Mg2+, were unable to reactivate rundown channels.7. Fluorescein 5-isothiocyanate (50 /uM), which interacts with lysine residues of the nucleotide-binding site on various ATPases, inhibited KATP channel activity. After wash-out, channel activity recovered only slightly. 8. These data suggest that the hydrolysis of ATP is important for reactivation of rundown KATP 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.

Hetero-concatemeric KIR6.X4/SUR14 Channels Display Distinct Conductivities but Uniform ATP Inhibition

“…K NDP channels were active in cell-attached patches, and the transition to a nucleotide-free intracellular solution in the inside-out configuration resulted in a transient increase in their activity before they closed. These K NDP channels remained in an operational state and could be activated by MgUDP, known to be an effective stimulator but a poor inhibitor of K IR 6.2/SUR1 channels (15), which does not reactivate run-down channels like MgATP (16)). The rapid wash-out of ATP applied in the presence of Mg 2ϩ resulted in a transient increase in K NDP channel activity similar to that observed upon transition to the inside-out configuration.…”

Anionic Phospholipids Activate ATP-sensitive Potassium Channels

“…Although Mg-ATP is not required to mediate activation by anionic phospholipids, the anionic lipid hypothesis can account for the well known Mg-ATP activation effect on KATPs. It has previously been reported that nonphosphorylating analogs of ATP, AMP-PNP and AMP-PCP, are incapable of increasing KATP activity, suggesting that Mg-ATP acts as a phosphoryl group donor in kinase-mediated phosphorylation reaction, although such kinases have not yet been identified (29). Our effectiveness se- The open times were fitted to two components of the exponential.…”

Reconstituted Human Cardiac K ATP Channels

“…47 We selected clearly striated cardiomyocytes with a length/width ratio of Ͼ4 and a resting membrane potential of ϷϪ70 mV (see Materials and Methods). Patches with channels displaying the mean PoϽ0.8 after patch isolation and/or reactivation by mmol/L MgATP, expected to refresh the channel maximally, 48 were discarded. For our expression studies, we used COSm6 cells that we have observed to have a negligible number of endogenous K IR , when compared with COS7, COS1, and HEK293 cells.…”