K_ATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP

Abstract

Single ATP-sensitive potassium channels (K _ATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated 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 μM; Li salt) reduced the open-probability p_o of K _ATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of p_o at −40 mV was observed at nucleotide concentrations of 29 μM in the absence and of 39 μM in the presence of Mg²⁺. The steepness of the AMP-PNP concentration-response 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 protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium 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 Li⁺ ions.