Dual effects of diazoxide on ATP‐K⁺ currents recorded from an insulin‐secreting cell line

Abstract

1 The effects of diazoxide on ATP-K+ channel currents, recorded from the insulin-secreting cell line, CRI-G1, were studied using patch-clamp techniques. 2 Under current-clamp recording conditions diazoxide (0.6 mM), inhibited action potential activity and hyperpolarized CRI-Gi cells with a concomitant increase in membrane conductance. Recordings from voltage-clamped whole-cells and isolated patches indicate that activation of ATP-K+ channel currents underlie these effects. 3 Diazoxide elicited an activation of ATP-K+ channels which had been partially inhibited by ATP, on application to either surface of the plasma membrane, although it was more effective when applied directly to the cytoplasmic side. Activation of the ATP-K+ currents involves an increase in the single channel open-state probability and an apparent increase in the number of functional channels. 4 Activation was observed only when Mg-ATP was present in the cytoplasmic bathing solution. There was no activation of currents by diazoxide when ATP, in the absence of Mg2+ ions, or Mg-AMP-PNP was present to inhibit the ATP-K+ channels. 5 In the absence of ATP and Mg2+ ions in the cytoplasmic bathing solution, diazoxide (0.6 mM) produced an inhibition of ATP-K + currents. 6 Cromakalim (BRL 34915) at 1OpM and 100pM had no significant effects on ATP-K+ currents. 7 It is concluded that diazoxide-induced activation of ATP-K + channel currents probably involves phosphorylation of the channel or some closely associated membrane protein.

The Novel Diazoxide Analog 3-Isopropylamino-7-Methoxy- 4H -1,2,4-Benzothiadiazine 1,1-Dioxide Is a Selective Kir6.2/SUR1 Channel Opener

“…5A compares the effect of 100 mol/l NNC 55-9216 in the presence of 100 mol/l MgATP, in the presence of 100 mol/l ATP but without Mg 2ϩ , and in the presence of 100 mol/l MgATP␥S. As has been observed for diazoxide (36,55), Mg 2ϩ was required for the ability of ATP to support channel activation by NNC 55-9216. Although this result is consistent with the idea that ATP hydrolysis is needed for NNC 55-9216 activation, we cannot exclude the alternative possibility that MgATP, but not ATP alone, is able to interact with the channel.…”

“…Interaction of nucleotides with the NBDs of SUR also modifies the response to PCOs. For example, stimulation of ␤-cell K ATP channels by diazoxide requires the presence of intracellular Mg nucleotide (36), and mutations in the NBDs of SUR1 abolish the ability of MgATP to support diazoxide activation (33,35). Likewise, although pinacidil can activate cardiac K ATP channels in the absence of added nucleotide (provided that the drug is tested before channel run-down is pronounced) (21,37,38), both the rate of deactivation of Kir6.2/SUR2A currents on removal of pinacidil and the ability to measure binding of the analog [ 3 H]P-1075 to SUR2A require the presence of MgATP and intact NBDs (38 -40).…”

Diazoxide‐sensitivity of the adenosine 5′‐triphosphate‐dependent K+ channel in mouse pancreatic β‐cells

“…It has been suggested that protein phosphorylation is involved in diazoxide-induced activation of the K-ATPchannels since hydrolysis of MgATP was necessary for channel activation by diazoxide (Dunne, 1989;Kozlowski et al, 1989). The inside-out experiments in our present study were carried out using an experimental protocol favouring protein phosphorylation.…”

“…Channel inhibition induced by occupation of the inhibitory receptor for cytosolic nucleotides is reversed by diazoxide (Trube et al, 1986;Dunne et al, 1987;Sturgess et al, 1988;Zunkler et al, 1988b;Kozlowski et al, 1989;Gillis et al, 1989;Dunne, 1989). As Ltd, 1992 cytosolic MgATP, it has been suggested that phosphorylation of the K-ATP-channel or a closely associated regulatory protein is involved in the mechanism of action of diazoxide (Kozlowski et al, 1989;Dunne, 1989).…”

Mg2+‐dependent inhibition of KATP by sulphonylureas in CRI‐G1 insulin‐secreting cells

“…Outward current (defined as the current flowing from the intra-to the extracellular side of the membrane) is shown as upward deflections on all traces. The single channel current analysis was determined off-line using a programme that incorporated a 50% threshold crossing parameter to detect events (Dempster, 1988) (Kozlowski et al, 1989;Kozlowski & Ashford, 1990 were applied at 2 s intervals as described previously (Sturgess et al, 1988;Kozlowski et al, 1989 In experiments using the inside-out configuration, the pi- …”