Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for 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, SUR2, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K(ATP) in low [ATP] (1 microM) 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 microM 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 microM and 5.8 mM, respectively. Phosphoinositides failed to desensitize adenosine inhibition of K(ATP). In the presence of SUR2, phosphoinositides attenuated MgATP antagonism of ATP inhibition. Kir6.2DeltaC35, a truncated Kir6.2 that functions without SUR2, 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.
Fan
Z
Z
Department of Physiology, University of Tennessee, College of Medicine, Memphis, Tennessee 38163, USA. zfan@physiol.utmem.edu
Makielski
J C
JC
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D019556:COS Cells; D004558:Electric Stimulation; D004594:Electrophysiology; D054086:KATP Channels; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D015335:Molecular Probes; D018408:Patch-Clamp Techniques; D010716:Phosphatidylinositols / Q000494:pharmacology*; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying
D018528:ATP-Binding Cassette Transporters; D054086:KATP Channels; D015335:Molecular Probes; D010716:Phosphatidylinositols; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C092520:uK-ATP-1 potassium channel; D000255:Adenosine Triphosphate; D006899:Mixed Function Oxygenases