STIMULATION OF THE KATP CHANNEL BY ADP AND DIAZOXIDE REQUIRES NUCLEOTIDE HYDROLYSIS IN MOUSE PANCREATIC BETA-CELLS

1. The mechanisms by which ADP and the hyperglycaemic compound diazoxide stimulate the activity of the ATP-regulated K+ channel (K(ATP) channel) were studied using inside-out patches isolated from mouse pancreatic beta-cells maintained in tissue culture. 2. The ability of diazoxide and ADP to increase K(ATP) channel activity declined with time following patch excision and no stimulation was observed after 15-40 min. 3. Activation of K(ATP) channels by ADP required the presence of intracellular Mg2+. The stimulatory effect of ADP was mimicked by AMP but only in the presence of ATP. Replacement of ATP with the non-hydrolysable analogue beta,gamma-methylene ATP did not interfere with the ability of ADP to stimulate K(ATP) channel activity. By contrast, enhancement of K(ATP) channel activity was critically dependent on hydrolysable ADP and no stimulation was observed after substitution of alpha,beta-methylene ADP for standard ADP. 4. The ability of diazoxide to enhance K(ATP) channel activity was dependent on the presence of both internal Mg2+ and ATP. Diazoxide stimulation of K(ATP) channel activity was not observed after substitution of beta,gamma-methylene ATP for ATP. However, in the presence of ADP, at a concentration which in itself had no stimulatory action (10 muM), diazoxide was stimulatory also in the presence of the stable ATP analogue. 5. The stimulatory action of diazoxide on K(ATP) channel activity in the presence of ATP was markedly enhanced by intracellular ADP. This potentiating effect of ADP was not reproduced by the stable analogue alpha,beta-methylene ADP and was conditional on the presence of intracellular Mg2+. A similar enhancement of channel activity was also observed with AMP (0.1 mm). In the absence of ATP, diazoxide was still capable of stimulating channel activity provided ADP was present. This effect was not reproduced by AMP. 6. In both nucleotide-free solution and in the presence of 0.1 mm ATP, the distribution of the K(ATP) channel open times were described by a single exponential with a time constant of almost-equal-to 20 ms. Addition of ADP or diazoxide resulted in the appearance of a second component with a time constant of > 100 ms which comprised 40-70% of the total number of events. Under the latter experimental conditions, the open probability of the channel increased more than fivefold relative to that observed in the presence of ATP alone. 7. We propose that ADP- and diazoxide-induced stimulation of K(ATP) channel activity reflects the same basal mechanism and involves a diffusible cytoplasmic regulatory component which is not part of the K(ATP) channel itself The effect of diazoxide is dependent on the presence of Mg-ADP and may result from amplification of the ADP action, culminating in the induction of a novel kinetic state characterized by openings of long duration.