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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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Mentions: The effects of PPIs on single-channel kinetics of KATP in the absence of ATP have been analyzed (Fan and Makielski 1997; this paper, Tables I–III). Mean open time was increased and mean closed time was decreased after treatment with PPIs requiring that PPIs affect a transition that is directly connected to open state(s). Given this consideration, a collateral change in ATP sensitivity secondary to change in maximal Po could be caused if ATP binds preferentially to closed states of the channel in a linear scheme (Fig. 1), where the arrows represent the direction toward which ATP or PPIs facilitate transition. Fig. 1 can qualitatively reproduce the burst behaviors we observed. If ATP binds only between the bursts, as when the channel is in C1 or C2, the probability that ATP binds to the channel would be reduced if PPIs increase burst duration and reduce the closed times between bursts. However, this scheme cannot explain the other kinetic changes we observed experimentally. In such a scheme, ATP should not affect open times, but ATP shortened open times, and PPIs altered the ATP effect on open times as well (Table and Table ). Interestingly, open times of Kir6.2ΔC35 had a similar [ATP] dependence (Table IV; and Drain et al. 1998; Trapp et al. 1998). Also, according to Fig. 1, the mean duration of the slowest component of closed intervals between bursts would be predicted to be dominated by [ATP], and PPIs should not affect this duration, but this was not the case (see τc,4 in Table ). Changes in both of these parameters are major contributors to the increase in channel activity in the presence of ATP after treatment with PPIs. Hence, the effect of PPIs on single-channel kinetics reflecting desensitization of ATP inhibition cannot be caused solely by interactions with ATP inhibitory closed states. In a recent study, Babenko et al. 1999a also reported that a model such as Fig. 1 could not account for the SUR influence on ATP inhibition and proposed that a true change in ATP affinity occurred.


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)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230641&req=5

Mentions: The effects of PPIs on single-channel kinetics of KATP in the absence of ATP have been analyzed (Fan and Makielski 1997; this paper, Tables I–III). Mean open time was increased and mean closed time was decreased after treatment with PPIs requiring that PPIs affect a transition that is directly connected to open state(s). Given this consideration, a collateral change in ATP sensitivity secondary to change in maximal Po could be caused if ATP binds preferentially to closed states of the channel in a linear scheme (Fig. 1), where the arrows represent the direction toward which ATP or PPIs facilitate transition. Fig. 1 can qualitatively reproduce the burst behaviors we observed. If ATP binds only between the bursts, as when the channel is in C1 or C2, the probability that ATP binds to the channel would be reduced if PPIs increase burst duration and reduce the closed times between bursts. However, this scheme cannot explain the other kinetic changes we observed experimentally. In such a scheme, ATP should not affect open times, but ATP shortened open times, and PPIs altered the ATP effect on open times as well (Table and Table ). Interestingly, open times of Kir6.2ΔC35 had a similar [ATP] dependence (Table IV; and Drain et al. 1998; Trapp et al. 1998). Also, according to Fig. 1, the mean duration of the slowest component of closed intervals between bursts would be predicted to be dominated by [ATP], and PPIs should not affect this duration, but this was not the case (see τc,4 in Table ). Changes in both of these parameters are major contributors to the increase in channel activity in the presence of ATP after treatment with PPIs. Hence, the effect of PPIs on single-channel kinetics reflecting desensitization of ATP inhibition cannot be caused solely by interactions with ATP inhibitory closed states. In a recent study, Babenko et al. 1999a also reported that a model such as Fig. 1 could not account for the SUR influence on ATP inhibition and proposed that a true change in ATP affinity occurred.

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