Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K+ channel -: A molecular probe or the mechanism of ATP-sensitive inhibition

Anionic phospholipids modulate the activity of inwardly rectifying potassium channels (Fan, Z., and J.C. Makielski. 1997. J. Biol. Chem. 272:5388-5395). The effect of phosphoinositides on adenosine triphosphate (ATP) inhibition of ATP-sensitive potassium channel (K-ATP,) currents was investigated using the inside-out patch clamp technique in cardiac myocytes and in COS-1 cells in which the cardiac isoform of the sulfonylurea receptor, SURF, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K-ATP, in low [ATP] (1 mu M) within 30 s. Phosphoinositides desensitized ATP inhibition with a longer onset period (>3 min), activating channels inhibited by ATP (1 mM). Phosphoinositides treatment for 10 min shifted the half-inhibitory [ATP] (K-i) from 35 mu M to 16 mM. At the single-channel level, increased [ATP] caused a shorter mean open time and a longer mean closed time. Phosphoinositides prolonged the mean open time, shortened the mean closed time, and weakened the [ATP] dependence of these parameters resulting in a higher open probability at any given [ATP]. The apparent rate constants for ATP binding were estimated to be 0.8 and 0.02 mM(-1) ms(-1) before and after 5-min treatment with phosphoinositides, which corresponds to a K-i of 35 mu M and 5.8 mM, respectively Phosphoinositides failed to desensitize adenosine inhibition of K-ATP, In the presence of SURF, phosphoinositides attenuated MgaTP antagonism of ATP inhibition. Kir6.2 Delta C35, a truncated Kir6.2 that functions without SURF, also exhibited phosphoinositide desensitization of ATP inhibition. These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition. We propose a model of the ATP binding site involving positively charged residues on the COOH-terminus of Kir6.2, with which phosphoinositides interact to desensitize ATP inhibition.