ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

Abstract

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10/zM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMPdependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500/~M in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in ldnase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 #M), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 #M, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

The mechanism of KATP channel inhibition by ATP.

“…This model explains why a gradual run-down of channel activity occurs at very low [ATP] (because there is no high energy phosphate to support phosphorylation), but also explains how rapid, reversible blockade by ATP and other nucleotides can be obtained. not compatible in any simple ~ way with the hypothesis of Ribalet et al (1989) and suggest that a different model may be needed to explain the channel behavior. Fig.…”

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Dear Sir, Ribalet et al (1989) have presented interesting data on the regulation of KAav channel activity by phosphorylation, and an intriguing hypothesis to explain KAy P channel inhibition by ATP. They propose that the opening and closing of the KAvp channel can be explained by a one-site phosphorylation model, outlined in Fig.

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“…1 A. According to the model of Ribalet et al (1989), the opening of the KAXp channel at low [ATP]i occurs as a result of channel phosphorylation by a cAMP-dependent protein kinase. At high [ATP]~ this kinase is inhibited by an ATP-dependent protein kinase inhibitor (PKI) so that background dephosphorylation leads to inhibition of channel activity.…”

“…We presume that each nucleotide acted in a similar manner to inhibit channel activity. According to the hypothesis of Ribalet et al (1989), channel activity would be decreased in each case because of reduced phosphorylation of the KAaV channel with continued background dephosphorylation. The critical observation with respect to the hypothesis being tested is the rapid reactivation of channel activity on switching to a nucleotide-free solution.…”

“…In each case a complete restoration of channel activity is seen when the patch is returned to the nucleotide-free solution. According to the hypothesis of Ribalet et al (1989), reopening of the KAVp channel should not occur in at least the first two of these experiments, because on exposure to a nucleotide-free solution (after having been exposed to AMP or AMP-PNP) the catalytic subunit will have AMP or AMP-PNP bound, and no high energy phosphate is available to reprime the catalytic subunit and rephosphorylate the channel. Fig.…”