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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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Two effects of PPIs on KATP currents. PPIs (1 mg/ml) and ATP (1 mM) were applied in the bath (internal membrane in these inside-out patches) at the times indicated by the bars. [ATP] was 1 μM unless indicated otherwise. The dashed line represents closed current levels. Currents were recorded from COS-1 cells transfected with Kir6.2/SUR2, and all three examples were multichannel patches. (A) Current recorded after run-down of KATP shows reactivation by PPIs. (B) Current recorded in the presence of ATP to block the channel shows loss of ATP sensitivity after PPIs. (C) Current recorded in the absence of obvious run-down shows that these channels also lost sensitivity to ATP, and this effect persisted after removal of PPIs from the bath.
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Figure 1: Two effects of PPIs on KATP currents. PPIs (1 mg/ml) and ATP (1 mM) were applied in the bath (internal membrane in these inside-out patches) at the times indicated by the bars. [ATP] was 1 μM unless indicated otherwise. The dashed line represents closed current levels. Currents were recorded from COS-1 cells transfected with Kir6.2/SUR2, and all three examples were multichannel patches. (A) Current recorded after run-down of KATP shows reactivation by PPIs. (B) Current recorded in the presence of ATP to block the channel shows loss of ATP sensitivity after PPIs. (C) Current recorded in the absence of obvious run-down shows that these channels also lost sensitivity to ATP, and this effect persisted after removal of PPIs from the bath.

Mentions: When applied to the cytoplasmic surface of excised patches, PPIs had two experimentally distinct activating effects: (i) they increased maximal KATP open probability (maximal Po, defined as the Po measured in the absence of inhibitory [ATP]) with an onset period <30 s; and (ii) they desensitized KATP to ATP inhibition with a longer onset period (>3 min). As commonly seen in excised patch experiments, open channel activity of KATP was reduced and eventually lost over several minutes in a process often called run-down. Fig. 1 A shows an example of PPIs applied to a patch containing run-down KATP channels. After the patch was excised, KATP were allowed to run down for ∼5 min in this experiment. With inhibitory concentrations of ATP absent from the solution, PPIs (1 mg/ml) subsequently applied to the inner side of the patch membrane rapidly (within 30 s) reactivated channel activity (similar results observed in 35 patches of dog heart cells, rat heart cells, and SUR2/Kir6.2). The channel activity persisted for many minutes after PPIs were removed from the bathing solution. We further investigated two aspects of this effect of PPIs. First, although run-down is, strictly speaking, an experimental phenomenon, it may have a common mechanism related to physiological regulatory processes involving endogenous phosphatidylinositol. With this in mind, we also examined the effect of PPIs in the absence of notable run-down. Absence of run-down was defined by a value not less than one for the ratio: maximal Po immediately before application of PPIs/maximal Po at the time of patch excision.


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)

Two effects of PPIs on KATP currents. PPIs (1 mg/ml) and ATP (1 mM) were applied in the bath (internal membrane in these inside-out patches) at the times indicated by the bars. [ATP] was 1 μM unless indicated otherwise. The dashed line represents closed current levels. Currents were recorded from COS-1 cells transfected with Kir6.2/SUR2, and all three examples were multichannel patches. (A) Current recorded after run-down of KATP shows reactivation by PPIs. (B) Current recorded in the presence of ATP to block the channel shows loss of ATP sensitivity after PPIs. (C) Current recorded in the absence of obvious run-down shows that these channels also lost sensitivity to ATP, and this effect persisted after removal of PPIs from the bath.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Two effects of PPIs on KATP currents. PPIs (1 mg/ml) and ATP (1 mM) were applied in the bath (internal membrane in these inside-out patches) at the times indicated by the bars. [ATP] was 1 μM unless indicated otherwise. The dashed line represents closed current levels. Currents were recorded from COS-1 cells transfected with Kir6.2/SUR2, and all three examples were multichannel patches. (A) Current recorded after run-down of KATP shows reactivation by PPIs. (B) Current recorded in the presence of ATP to block the channel shows loss of ATP sensitivity after PPIs. (C) Current recorded in the absence of obvious run-down shows that these channels also lost sensitivity to ATP, and this effect persisted after removal of PPIs from the bath.
Mentions: When applied to the cytoplasmic surface of excised patches, PPIs had two experimentally distinct activating effects: (i) they increased maximal KATP open probability (maximal Po, defined as the Po measured in the absence of inhibitory [ATP]) with an onset period <30 s; and (ii) they desensitized KATP to ATP inhibition with a longer onset period (>3 min). As commonly seen in excised patch experiments, open channel activity of KATP was reduced and eventually lost over several minutes in a process often called run-down. Fig. 1 A shows an example of PPIs applied to a patch containing run-down KATP channels. After the patch was excised, KATP were allowed to run down for ∼5 min in this experiment. With inhibitory concentrations of ATP absent from the solution, PPIs (1 mg/ml) subsequently applied to the inner side of the patch membrane rapidly (within 30 s) reactivated channel activity (similar results observed in 35 patches of dog heart cells, rat heart cells, and SUR2/Kir6.2). The channel activity persisted for many minutes after PPIs were removed from the bathing solution. We further investigated two aspects of this effect of PPIs. First, although run-down is, strictly speaking, an experimental phenomenon, it may have a common mechanism related to physiological regulatory processes involving endogenous phosphatidylinositol. With this in mind, we also examined the effect of PPIs in the absence of notable run-down. Absence of run-down was defined by a value not less than one for the ratio: maximal Po immediately before application of PPIs/maximal Po at the time of patch excision.

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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Related in: MedlinePlus