K-ATP CHANNELS OF MOUSE SKELETAL-MUSCLE - MECHANISM OF CHANNEL BLOCKAGE BY AMP-PNP

Single ATP-sensitive potassium channels (K-ATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Cha nnel blockage by the non-hydrolysable ATP analogue AMP-PNP was investi gated in the absence or presence of 1 mM MgCl2 with K+-rich solutions bathing the internal membrane surface. Currents through single. K-ATP channels were recorded at -40 and +40 mV. AMP-PNP (5 to 500 mu M; Li s alt) reduced the open-probability p(o) of K-ATP channels and decreased the single-channel currents at high nucleotide concentrations by appr oximately 10%. Half maximal reduction of p, at -40 mV was observed at nucleotide concentrations of 29 mu M in the absence and of 39 mu M in the presence of Mg2+. The steepness of the AMP-PNP concentration-respo nse curves was strongly affected by Mg2+, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl2. The efficacies of channel blockage by AMP-PNP at -40 and +40 mV were not significantly different. The results indicate that a K-ATP channel can bind more divalent Mg2+-complexes of AMP-PNP than trivalent proto nated forms of the nucleotide and that channel blockage is hardly affe cted by the membrane electric field. To estimate the contribution of l ithium ions to the observed results, we studied the effects of LiCl (0 .8 to 10 mM) in the Mg2+-free solution on the single channel current i . At a Li+ concentration of 10 mM, i was hardly affected at -40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of K-ATP channels by internal Liions.