On the mechanism of ADP-induced alteration of sulphonylurea sensitivity in cardiac ATP-sensitive K(+) channels

Abstract

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 tolbutamide inhibition of K(ATP) channels (IC(50)) 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 IC(50) from 0.4 to 5.3 and 11.4 mM, respectively. Increasing the ADP concentration further to 1 and 3 mM conversely reduced the IC(50) to 9.5 and 4.4 mM, respectively. 4. In the absence of Mg(2+) and ADP, the IC(50) was calculated to 16.6 mM which was found to be less, 12.3, 5.1 and 2.5 mM, respectively, when the ADP concentration 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 Mg(2+) 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 site that increases such sensitivity. Dissociation constants calculated for ADP to sites 1 and 2 were 2.6 microM and 46.7 mM, respectively. In the absence of Mg(2+), data were fitted by equations corresponding to a single site model (site 2); the dissociation constant for ADP was 25.0 mM. 6. It is concluded that ADP modifies tolbutamide sensitivity by binding to two sites. The high affinity site is strongly Mg(2+)-dependent, whereas the low affinity site is Mg(2+)-independent.

Figure 1

Effects of ADP on the tolbutamide-induced inhibition of K_ATP channels. (A) ADP lowers the efficacy of tolbutamide blocking of the channel. The fractional K_ATP channel current recorded from the inside-out membrane patch is illustrated. Arrows indicate the zero current levels. The bars shown above each trace indicate the period of exposure of membrane patches to the test solution containing tolbutamide (tolb) or ADP in the absence of ATP. The solution containing 0.3 mM ATP was superfused at the beginning and the end of traces to prevent the rundown of the channel activity. The activity of K_ATP channel is immediately increased and decreased at the time when ATP in the solution was removed and resuperfused, respectively. (B) Continuous recordings were made to test the effects of different concentrations of ADP (1 and 3 mM) on the blocking efficacy of 10 mM tolbutamide. (C) Concentration-inhibition relationship of activity of K_ATP channels for tolbutamide obtained during superfusion of various concentrations of ADP.

Figure 2

Effects of ADP on the tolbutamide-induced inhibition of K_ATP channels in the absence of Mg²⁺. (A) ADP enhanced the efficacy of tolbutamide blocking of the channel. The current fractions illustrated were recorded from the same membrane patch and filtered at 800 Hz. The bars shown above each trace indicate the period of exposure of the membrane patch to the test solution containing tolbutamide or ADP in the absence of ATP. A solution containing 0.3 mM ATP was superfused at the beginning and end of traces to protect the channel activity from rundown. (B) Concentration-inhibition relationships obtained during superfusion with various concentrations of ADP.

Figure 3

Effects of ATP on the tolbutamide sensitivity of K_ATP channels in the presence of Mg²⁺. (A) The bars above each trace indicate the period of exposure of membrane patches to the test solution. In the lower trace, 100 μM tolbutamide applied during the period indicated by the bar in the absence of ATP showed little effect on the channel activity, whereas it decreased the activity in the presence of 0.1 mM ATP (lower trace). (B) Concentration-inhibition relationship between tolbutamide concentration and channel activity is shown in the absence and presence of 0.1 mM ATP.

Figure 4

Changes in IC₅₀ for tolbutamide inhibition obtained during exposure to various concentrations of ADP in the presence and absence of Mg²⁺. The lines in the presence and absence of Mg²⁺ were plotted according to the equation (3) and equation (6), respectively. The error bars indicate the 95% confidence intervals of each data point.