Ribalet B, Ciani S, Eddlestone G T
Department of Physiology, Ahmanson Laboratory of Neurobiology, University of California, Los Angeles 90024.
J Gen Physiol. 1989 Oct;94(4):693-717. doi: 10.1085/jgp.94.4.693.
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 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent 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 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase 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 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, 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.
采用单通道记录技术研究胰岛素分泌细胞中赋予代谢物敏感钾通道ATP敏感性的机制。ATP在0 - 10微摩尔范围内刺激通道活性，但在更高浓度时则抑制该活性。在膜内面向外的膜片中，添加cAMP依赖性蛋白激酶抑制剂（PKI）可降低通道活性，这表明ATP的刺激作用是通过cAMP依赖性蛋白激酶介导的磷酸化作用实现的。在存在外源性PKI的情况下，将ATP浓度提高到10至500微摩尔可逐渐降低通道活性；据推测，这种失活是由于PKI或具有类似抑制特性的蛋白质与激酶的ATP依赖性结合导致激酶活性降低所致。开发了一个描述ATP作用的模型，该模型纳入了核苷酸的这两种不同作用。假设ATP控制通道活性的效力取决于与膜相关的抑制剂和催化亚基的相对浓度，我们的模型预测，当这两种调节剂的比例改变时，通道对ATP的敏感性将会变化。基于此，研究表明，在切除的膜片中通道对低ATP浓度的敏感性与细胞内ATP水平升高之间存在的明显差异，可以通过假设由于膜片切除后蛋白激酶的丧失导致抑制剂/激酶比例从1:1变为3:2来解释。在低浓度ATP（10 - 20微摩尔）下，一种不可水解的ATP类似物AMP - PNP在浓度低于10微摩尔时增强通道活性，而在较高浓度时则阻断通道活性。据推测，在前一种情况下AMP - PNP抑制激酶 - 抑制剂复合物的形成，而在后一种情况下阻止磷酸转移。类似的机制可以解释ATP与ADP之间的相互作用，其特征是在低ADP浓度下活性增强而在高浓度下阻断。
