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Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Fan Z, Makielski JC - J. Gen. Physiol. (1999)

Bottom Line: Biol.Phosphoinositides failed to desensitize adenosine inhibition of K(ATP).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee, College of Medicine, Memphis, Tennessee 38163, USA. zfan@physiol.utmem.edu

ABSTRACT
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.

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Plot of the reciprocal of the corrected mean open time (to) against [ATP] before (•) and after treatment with PPIs (5 min, 1 mg/ml) (○). (A) Data from rat ventricular myocytes (Table ). The lines are fits to  with inhibition rate constants of 0.8 and 0.02 mM−1 ms−1 for Kis of 35 μM and 5.8 mM for control and after treatment with PPIs, respectively. (B) Data for Kir6.2ΔC35 channels expressed in COS-1 cells converted from Table . The lines are obtained by taking an inhibition rate constant of 0.6 and 0.3 mM−1 ms−1 for Kis of 247 μM and 2.1 mM for control and after treatment with PPIs, respectively.
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Figure 6: Plot of the reciprocal of the corrected mean open time (to) against [ATP] before (•) and after treatment with PPIs (5 min, 1 mg/ml) (○). (A) Data from rat ventricular myocytes (Table ). The lines are fits to with inhibition rate constants of 0.8 and 0.02 mM−1 ms−1 for Kis of 35 μM and 5.8 mM for control and after treatment with PPIs, respectively. (B) Data for Kir6.2ΔC35 channels expressed in COS-1 cells converted from Table . The lines are obtained by taking an inhibition rate constant of 0.6 and 0.3 mM−1 ms−1 for Kis of 247 μM and 2.1 mM for control and after treatment with PPIs, respectively.

Mentions: Mean open time, to, varied with [ATP] both with and without PPIs (Table ), indicating that the ATP-inhibited state is an open state(s). Davies et al. 1992 used a method to obtain apparent ATP inhibition rate constants that did not depend upon other details of the underlying kinetic model. If we assume that (a) ATP binds to the open states to inhibit the channel; (b) rates of all ATP-dependent transitions linked to the open states are uniform and independent each other; and (c) ATP does not affect the relative occupancy within the set of open states, then the reciprocal of to has a linear relation to [ATP] given by: 4\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{1}/{t_{{\mathrm{o}}}}= \left \left({1}/{t_{{\mathrm{o}}}}\right) \right _{ \left \left( \left \left[{\mathrm{ATP}}\right] \right =0\right) \right }+r_{1} \left \left[{\mathrm{ATP}}\right] \right {\mathrm{,}}\end{equation*}\end{document} where the slope r1 will also be the apparent association rate constant for ATP causing inhibition. In the absence of evidence disqualifying these assumptions, we have accepted them for this analysis but use the term apparent affinity and apparent rate constants to acknowledge that state-dependent interactions may also play a role (Shyng and Nichols 1998). Fig. 6 A compares the relationship between [ATP] and 1/to with and without PPIs in rat ventricular myocytes. The linear fit both cases well, but untreated channels demonstrated a steeper slope (0.8 mM−1 ms−1) than channels treated with PPIs (0.02 mM−1 ms−1). Multiplying these slopes by the Ki (35 μM and 5.8 mM, determined earlier) gives dissociation rate constants of 0.03 and 0.12 ms−1 for channels untreated and treated with PPIs, respectively. Considering the proposed tetrameric stoichiometry of KATP, the actual inhibition and dissociation constants should be one-fourth of these values if ATP binds to any of four monomers to induce inhibition. Thus, at the single-channel level, PPIs both decreased the apparent ATP association rate and increased the apparent ATP dissociation rate for KATP.


Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Fan Z, Makielski JC - J. Gen. Physiol. (1999)

Plot of the reciprocal of the corrected mean open time (to) against [ATP] before (•) and after treatment with PPIs (5 min, 1 mg/ml) (○). (A) Data from rat ventricular myocytes (Table ). The lines are fits to  with inhibition rate constants of 0.8 and 0.02 mM−1 ms−1 for Kis of 35 μM and 5.8 mM for control and after treatment with PPIs, respectively. (B) Data for Kir6.2ΔC35 channels expressed in COS-1 cells converted from Table . The lines are obtained by taking an inhibition rate constant of 0.6 and 0.3 mM−1 ms−1 for Kis of 247 μM and 2.1 mM for control and after treatment with PPIs, respectively.
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Related In: Results  -  Collection

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Figure 6: Plot of the reciprocal of the corrected mean open time (to) against [ATP] before (•) and after treatment with PPIs (5 min, 1 mg/ml) (○). (A) Data from rat ventricular myocytes (Table ). The lines are fits to with inhibition rate constants of 0.8 and 0.02 mM−1 ms−1 for Kis of 35 μM and 5.8 mM for control and after treatment with PPIs, respectively. (B) Data for Kir6.2ΔC35 channels expressed in COS-1 cells converted from Table . The lines are obtained by taking an inhibition rate constant of 0.6 and 0.3 mM−1 ms−1 for Kis of 247 μM and 2.1 mM for control and after treatment with PPIs, respectively.
Mentions: Mean open time, to, varied with [ATP] both with and without PPIs (Table ), indicating that the ATP-inhibited state is an open state(s). Davies et al. 1992 used a method to obtain apparent ATP inhibition rate constants that did not depend upon other details of the underlying kinetic model. If we assume that (a) ATP binds to the open states to inhibit the channel; (b) rates of all ATP-dependent transitions linked to the open states are uniform and independent each other; and (c) ATP does not affect the relative occupancy within the set of open states, then the reciprocal of to has a linear relation to [ATP] given by: 4\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{1}/{t_{{\mathrm{o}}}}= \left \left({1}/{t_{{\mathrm{o}}}}\right) \right _{ \left \left( \left \left[{\mathrm{ATP}}\right] \right =0\right) \right }+r_{1} \left \left[{\mathrm{ATP}}\right] \right {\mathrm{,}}\end{equation*}\end{document} where the slope r1 will also be the apparent association rate constant for ATP causing inhibition. In the absence of evidence disqualifying these assumptions, we have accepted them for this analysis but use the term apparent affinity and apparent rate constants to acknowledge that state-dependent interactions may also play a role (Shyng and Nichols 1998). Fig. 6 A compares the relationship between [ATP] and 1/to with and without PPIs in rat ventricular myocytes. The linear fit both cases well, but untreated channels demonstrated a steeper slope (0.8 mM−1 ms−1) than channels treated with PPIs (0.02 mM−1 ms−1). Multiplying these slopes by the Ki (35 μM and 5.8 mM, determined earlier) gives dissociation rate constants of 0.03 and 0.12 ms−1 for channels untreated and treated with PPIs, respectively. Considering the proposed tetrameric stoichiometry of KATP, the actual inhibition and dissociation constants should be one-fourth of these values if ATP binds to any of four monomers to induce inhibition. Thus, at the single-channel level, PPIs both decreased the apparent ATP association rate and increased the apparent ATP dissociation rate for KATP.

Bottom Line: Biol.Phosphoinositides failed to desensitize adenosine inhibition of K(ATP).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee, College of Medicine, Memphis, Tennessee 38163, USA. zfan@physiol.utmem.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus