On the mechanism of ADP-induced alteration of sulphonylurea sensitivity incardiac ATP-sensitive K+ channels

1 To study the mechanism of regulation of sulphonylurea sensitivity in ATP- sensitive K+ (K-ATP) channels, we used the inside-out patch clamp technique in guinea-pig ventricular myocytes. 2 In the absence of nucleotides, the half maximal concentration of tolbutam ide inhibition of K-ATP channels (IC50) was 0.4 mM, and it decreased to 0.1 mM when 0.1 mM ATP was added. 3 Increasing the ADP concentration from 0 to 0.1 and 0.3 mM in the absence of ATP shifted the IC50 from 0.4 to 5.3 and 11.4 mM, respectively. Increasi ng the ADP concentration further to 1 and 3 mM conversely reduced the IC50 to 9.5 and 4.3 mM, respectively. 4 In the absence of Mg2+ and ADP, the IC50 was calculated to 16.6 mM which was found to be less, 12.3, 5.1 and 2.5 mM, respectively, when the ADP conc entration was increased to 0.1, 0.3 and 1 mM. 5 The IC(50)s for tolbutamide obtained at various concentrations of ADP in the presence of Mg2+ were best fitted by equations reflecting a model that assumed two binding sites for ADP; one is a high affinity site that reduces the sensitivity to the sulphonylurea, while the other is a low affinity si te that increases such sensitivity. Dissociation constants calculated for A DP to sites 1 and 2 were 2.6 mu M and 46.7 mM, respectively. In the absence of Mg2+, data were fitted by equations corresponding to a single site mode l (site 2); the dissociation constant for ADP was 25.0 mM. 6 It is concluded that ADP modifies tolbutamide sensitivity by binding to t wo sites. The high affinity site is strongly Mg2+-dependent, whereas the lo w affinity site is Mg2+-independent.