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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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Effect of adenosine on KATP before and after treatment with PPIs. (A) KATP currents recorded from a ventricular myocyte of rat heart. Current traces on the left were recorded after a 20-s perfusion of PPIs (1 mg/ml). ATP and adenosine were added at the concentrations shown. Traces on the right were recorded after 10-min treatment with PPIs (1 mg/ml). c– indicates the closed current level Traces were recorded from a single patch, and at least six channels were present in this patch. (B) Effect of adenosine on current from a COS-1 cell expressing Kir6.2ΔC35. (a) Current traces were recorded before (left) and after (right) treatment for 10 min with PPIs (1 mg/ml). c– indicates the current level where all channels were closed. Traces were recorded from a single patch, and at least five channels were present in this patch. (b) Amplitude histogram calculated from the corresponding currents in A (see text for details).
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Figure 9: Effect of adenosine on KATP before and after treatment with PPIs. (A) KATP currents recorded from a ventricular myocyte of rat heart. Current traces on the left were recorded after a 20-s perfusion of PPIs (1 mg/ml). ATP and adenosine were added at the concentrations shown. Traces on the right were recorded after 10-min treatment with PPIs (1 mg/ml). c– indicates the closed current level Traces were recorded from a single patch, and at least six channels were present in this patch. (B) Effect of adenosine on current from a COS-1 cell expressing Kir6.2ΔC35. (a) Current traces were recorded before (left) and after (right) treatment for 10 min with PPIs (1 mg/ml). c– indicates the current level where all channels were closed. Traces were recorded from a single patch, and at least five channels were present in this patch. (b) Amplitude histogram calculated from the corresponding currents in A (see text for details).

Mentions: To test the hypothesis that charged lipid–protein interactions play a role in the effect of PPIs on ATP sensitivity, we probed the mechanism of inhibition by testing uncharged adenosine to represent the uncharged adenosine moiety of ATP. Fig. 9 A shows an example of the experiments with adenosine before and after a 10-min treatment with PPIs. Note that in this example the control was taken after a 20-s period of perfusion with PPIs. This brief treatment with PPIs induced little or no change in ATP sensitivity; ATP inhibition had a Ki (37 μM measured in one patch) not significantly different from the value obtained without any treatment with PPIs (Fig. 2). Adenosine (1 mM) produced a moderate inhibition (∼20% of maximal Po,). After a 10-min treatment with PPIs, ATP sensitivity was profoundly reduced, whereas the inhibitory effect of adenosine was much less affected. We repeated the same experiments in Kir6.2ΔC35. Fig. 9 B a shows current recordings from a typical experiment. This patch contained at least five active channels. Because of the short openings typical of Kir6.2ΔC35, macroscopic currents with this number of channels present appear very noisy. Figure 9 B b better demonstrates the current levels using a histogram transform at a current resolution (bin width) of 0.05 pA. Each peak of the histogram represents an open channel level. The statistical values of Po at different [ATP] and [adenosine] relative to the maximal Po measured at 1 μM ATP in the same preparations are given in Fig. 10. We noticed that adenosine also produced less inhibition in Kir6.2ΔC35 than in KATP of rat ventricular myocytes. The statistical data also demonstrated that treatment with PPIs significantly attenuated ATP inhibition, but PPIs had little effect on adenosine inhibition.


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)

Effect of adenosine on KATP before and after treatment with PPIs. (A) KATP currents recorded from a ventricular myocyte of rat heart. Current traces on the left were recorded after a 20-s perfusion of PPIs (1 mg/ml). ATP and adenosine were added at the concentrations shown. Traces on the right were recorded after 10-min treatment with PPIs (1 mg/ml). c– indicates the closed current level Traces were recorded from a single patch, and at least six channels were present in this patch. (B) Effect of adenosine on current from a COS-1 cell expressing Kir6.2ΔC35. (a) Current traces were recorded before (left) and after (right) treatment for 10 min with PPIs (1 mg/ml). c– indicates the current level where all channels were closed. Traces were recorded from a single patch, and at least five channels were present in this patch. (b) Amplitude histogram calculated from the corresponding currents in A (see text for details).
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Related In: Results  -  Collection

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Figure 9: Effect of adenosine on KATP before and after treatment with PPIs. (A) KATP currents recorded from a ventricular myocyte of rat heart. Current traces on the left were recorded after a 20-s perfusion of PPIs (1 mg/ml). ATP and adenosine were added at the concentrations shown. Traces on the right were recorded after 10-min treatment with PPIs (1 mg/ml). c– indicates the closed current level Traces were recorded from a single patch, and at least six channels were present in this patch. (B) Effect of adenosine on current from a COS-1 cell expressing Kir6.2ΔC35. (a) Current traces were recorded before (left) and after (right) treatment for 10 min with PPIs (1 mg/ml). c– indicates the current level where all channels were closed. Traces were recorded from a single patch, and at least five channels were present in this patch. (b) Amplitude histogram calculated from the corresponding currents in A (see text for details).
Mentions: To test the hypothesis that charged lipid–protein interactions play a role in the effect of PPIs on ATP sensitivity, we probed the mechanism of inhibition by testing uncharged adenosine to represent the uncharged adenosine moiety of ATP. Fig. 9 A shows an example of the experiments with adenosine before and after a 10-min treatment with PPIs. Note that in this example the control was taken after a 20-s period of perfusion with PPIs. This brief treatment with PPIs induced little or no change in ATP sensitivity; ATP inhibition had a Ki (37 μM measured in one patch) not significantly different from the value obtained without any treatment with PPIs (Fig. 2). Adenosine (1 mM) produced a moderate inhibition (∼20% of maximal Po,). After a 10-min treatment with PPIs, ATP sensitivity was profoundly reduced, whereas the inhibitory effect of adenosine was much less affected. We repeated the same experiments in Kir6.2ΔC35. Fig. 9 B a shows current recordings from a typical experiment. This patch contained at least five active channels. Because of the short openings typical of Kir6.2ΔC35, macroscopic currents with this number of channels present appear very noisy. Figure 9 B b better demonstrates the current levels using a histogram transform at a current resolution (bin width) of 0.05 pA. Each peak of the histogram represents an open channel level. The statistical values of Po at different [ATP] and [adenosine] relative to the maximal Po measured at 1 μM ATP in the same preparations are given in Fig. 10. We noticed that adenosine also produced less inhibition in Kir6.2ΔC35 than in KATP of rat ventricular myocytes. The statistical data also demonstrated that treatment with PPIs significantly attenuated ATP inhibition, but PPIs had little effect on adenosine inhibition.

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