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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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Effects of PPIs and Mg2+ on ATP inhibition. (A) Current traces recorded in a patch excised from a rat ventricular myocyte before (Control) and after 5-min treatment with PPIs (0.5 mg/ml) (labeled as PPIs). The dotted lines indicate unitary channel current levels. The unitary channel current level in MgCl2 2.2 mM was scaled up to the same level in Mg 0 to assist in making the comparison. c– denotes the closed level. In control, 0.5 mM ATP inhibited KATP (middle trace), but this inhibition was antagonized by MgCl2 2.2 mM (right trace). After treatment with PPIs, more channels were open in low [ATP], 0.5 mM was less effective in suppressing KATP, and MgCl2 2.2 mM slightly antagonized ATP inhibition. (B) Summary data for effects of PPIs and MgCl2 2.2 mM on ATP sensitivity were fitted as described for Fig. 2. The symbols represent the mean ± SE for nine patches. Before treatment with PPIs, Ki = 30 ± 2.8 μM in 0 Mg (•) and 333 ± 56 μM in 2.2 mM Mg (○) (P < 0.001). After treatment with PPIs, Ki = 2.3 ± 0.4 mM in 0 Mg (▴) and 3.4 ± 0.2 mM in 2.2 mM Mg (▵) (P = 0.03).
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Figure 3: Effects of PPIs and Mg2+ on ATP inhibition. (A) Current traces recorded in a patch excised from a rat ventricular myocyte before (Control) and after 5-min treatment with PPIs (0.5 mg/ml) (labeled as PPIs). The dotted lines indicate unitary channel current levels. The unitary channel current level in MgCl2 2.2 mM was scaled up to the same level in Mg 0 to assist in making the comparison. c– denotes the closed level. In control, 0.5 mM ATP inhibited KATP (middle trace), but this inhibition was antagonized by MgCl2 2.2 mM (right trace). After treatment with PPIs, more channels were open in low [ATP], 0.5 mM was less effective in suppressing KATP, and MgCl2 2.2 mM slightly antagonized ATP inhibition. (B) Summary data for effects of PPIs and MgCl2 2.2 mM on ATP sensitivity were fitted as described for Fig. 2. The symbols represent the mean ± SE for nine patches. Before treatment with PPIs, Ki = 30 ± 2.8 μM in 0 Mg (•) and 333 ± 56 μM in 2.2 mM Mg (○) (P < 0.001). After treatment with PPIs, Ki = 2.3 ± 0.4 mM in 0 Mg (▴) and 3.4 ± 0.2 mM in 2.2 mM Mg (▵) (P = 0.03).

Mentions: In the presence of Mg2+, the potency of ATP inhibition of KATP is partially reduced, an effect attributed to MgATP stimulation of KATP through interaction with the SUR subunit (Gribble et al. 1998). Much like PPIs, the effect of the presence of Mg2+ shifts the ATP concentration–inhibition curve to the right. Both Mg2+ and PPIs are potentially cellular regulators. Therefore, from both physiologic and mechanistic points of view, it is important to know whether PPIs and MgATP effects on ATP inhibition are simply additive or interactive (synergistic or antagonistic). ATP inhibition of KATP was reduced in the presence of MgCl2 2.2 mM both before and after treatment with PPIs (Fig. 3 A), but the change in ATP inhibition was much less dramatic after treatment. Summary data (Fig. 3 B) show that before treatment with PPIs, ATP inhibition in the presence of Mg2+ decreased by ∼10-fold, whereas after treatment with PPIs, the same [Mg2+] caused only a 1.5-fold decrease in ATP inhibition. For this experiment, we used a fixed [MgCl2] for all [ATP] to avoid the additional errors introduced by titration of free [Mg2+]. However, this meant that the [Mg2+] was likely to be reduced at the higher [ATP] and could have had a reduced effect. We therefore chose a 5-min exposure to 0.5 mg/ml PPIs, which gave a Ki of 2.3 mM, much less than the 16 mM obtained with longer exposures (Fig. 2 C). Under this nonsaturating condition we were able to record the proportional changes of KATP activity over a common [ATP] range, mitigating the possible problem caused by comparing channel activity at different [Mg2+]. In addition, in 2 mM of ATP, the highest [ATP] we used, doubling [MgCl2] to 4.4 mM did not significantly change Po (data not shown). Therefore, we conclude that the Mg2+ effect is not strictly additive to the effect of PPIs and that the two effects interfere each other, and likely share a linked mechanism.


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)

Effects of PPIs and Mg2+ on ATP inhibition. (A) Current traces recorded in a patch excised from a rat ventricular myocyte before (Control) and after 5-min treatment with PPIs (0.5 mg/ml) (labeled as PPIs). The dotted lines indicate unitary channel current levels. The unitary channel current level in MgCl2 2.2 mM was scaled up to the same level in Mg 0 to assist in making the comparison. c– denotes the closed level. In control, 0.5 mM ATP inhibited KATP (middle trace), but this inhibition was antagonized by MgCl2 2.2 mM (right trace). After treatment with PPIs, more channels were open in low [ATP], 0.5 mM was less effective in suppressing KATP, and MgCl2 2.2 mM slightly antagonized ATP inhibition. (B) Summary data for effects of PPIs and MgCl2 2.2 mM on ATP sensitivity were fitted as described for Fig. 2. The symbols represent the mean ± SE for nine patches. Before treatment with PPIs, Ki = 30 ± 2.8 μM in 0 Mg (•) and 333 ± 56 μM in 2.2 mM Mg (○) (P < 0.001). After treatment with PPIs, Ki = 2.3 ± 0.4 mM in 0 Mg (▴) and 3.4 ± 0.2 mM in 2.2 mM Mg (▵) (P = 0.03).
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Figure 3: Effects of PPIs and Mg2+ on ATP inhibition. (A) Current traces recorded in a patch excised from a rat ventricular myocyte before (Control) and after 5-min treatment with PPIs (0.5 mg/ml) (labeled as PPIs). The dotted lines indicate unitary channel current levels. The unitary channel current level in MgCl2 2.2 mM was scaled up to the same level in Mg 0 to assist in making the comparison. c– denotes the closed level. In control, 0.5 mM ATP inhibited KATP (middle trace), but this inhibition was antagonized by MgCl2 2.2 mM (right trace). After treatment with PPIs, more channels were open in low [ATP], 0.5 mM was less effective in suppressing KATP, and MgCl2 2.2 mM slightly antagonized ATP inhibition. (B) Summary data for effects of PPIs and MgCl2 2.2 mM on ATP sensitivity were fitted as described for Fig. 2. The symbols represent the mean ± SE for nine patches. Before treatment with PPIs, Ki = 30 ± 2.8 μM in 0 Mg (•) and 333 ± 56 μM in 2.2 mM Mg (○) (P < 0.001). After treatment with PPIs, Ki = 2.3 ± 0.4 mM in 0 Mg (▴) and 3.4 ± 0.2 mM in 2.2 mM Mg (▵) (P = 0.03).
Mentions: In the presence of Mg2+, the potency of ATP inhibition of KATP is partially reduced, an effect attributed to MgATP stimulation of KATP through interaction with the SUR subunit (Gribble et al. 1998). Much like PPIs, the effect of the presence of Mg2+ shifts the ATP concentration–inhibition curve to the right. Both Mg2+ and PPIs are potentially cellular regulators. Therefore, from both physiologic and mechanistic points of view, it is important to know whether PPIs and MgATP effects on ATP inhibition are simply additive or interactive (synergistic or antagonistic). ATP inhibition of KATP was reduced in the presence of MgCl2 2.2 mM both before and after treatment with PPIs (Fig. 3 A), but the change in ATP inhibition was much less dramatic after treatment. Summary data (Fig. 3 B) show that before treatment with PPIs, ATP inhibition in the presence of Mg2+ decreased by ∼10-fold, whereas after treatment with PPIs, the same [Mg2+] caused only a 1.5-fold decrease in ATP inhibition. For this experiment, we used a fixed [MgCl2] for all [ATP] to avoid the additional errors introduced by titration of free [Mg2+]. However, this meant that the [Mg2+] was likely to be reduced at the higher [ATP] and could have had a reduced effect. We therefore chose a 5-min exposure to 0.5 mg/ml PPIs, which gave a Ki of 2.3 mM, much less than the 16 mM obtained with longer exposures (Fig. 2 C). Under this nonsaturating condition we were able to record the proportional changes of KATP activity over a common [ATP] range, mitigating the possible problem caused by comparing channel activity at different [Mg2+]. In addition, in 2 mM of ATP, the highest [ATP] we used, doubling [MgCl2] to 4.4 mM did not significantly change Po (data not shown). Therefore, we conclude that the Mg2+ effect is not strictly additive to the effect of PPIs and that the two effects interfere each other, and likely share a linked mechanism.

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