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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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PPIs reactivate KATP current in Kir6.2ΔC35. Single-channel currents were recorded from an inside-out patch from a COS-1 cell transfected with Kir6.2ΔC35. c– indicates the closed open level. The bottom trace in each panel represents a detail at a greater time resolution of the record in the top panel. The diagonal lines indicate the part of the top record that was selected for display in the lower record. (A) KATP current recorded immediately after excision of the patch before run-down. (B) Current recorded 10 min after excision of the patch after the current had run down. (C) Current recorded after a 5-min treatment with PPIs (1 mg/ml).
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Figure 7: PPIs reactivate KATP current in Kir6.2ΔC35. Single-channel currents were recorded from an inside-out patch from a COS-1 cell transfected with Kir6.2ΔC35. c– indicates the closed open level. The bottom trace in each panel represents a detail at a greater time resolution of the record in the top panel. The diagonal lines indicate the part of the top record that was selected for display in the lower record. (A) KATP current recorded immediately after excision of the patch before run-down. (B) Current recorded 10 min after excision of the patch after the current had run down. (C) Current recorded after a 5-min treatment with PPIs (1 mg/ml).

Mentions: Which subunit, SUR2 or Kir6.2, interacts with PPIs to cause the antagonism of ATP inhibition? Previously, using site-directed mutagenesis, we have shown that the reactivation effect of PPIs on KATP was through an interaction on the Kir6.2 subunit (Fan and Makielski 1997). Moreover, phosphatidylinositol is known to have a direct interaction with other inwardly rectifying K+ channels (Fan and Makielski 1997; Huang et al. 1998). Therefore, we explored whether the ATP desensitization effect of PPIs also localized to the channel subunit. When Kir6.2 was expressed alone, either with or without a green florescence protein tag on its COOH terminus (John et al. 1998), or with COOH-terminal truncation (Tucker et al. 1997), Kir6.2 retained ATP sensitivity, albeit reduced. We examined the effect of PPIs on ATP inhibition of the COOH-terminal truncated Kir6.2, Kir6.2ΔC35, that when expressed alone without SUR2 gave KATP currents. In the presence of low [ATP] (1 μM), the gating behavior of Kir6.2ΔC35 was characterized by short openings and less obvious short bursts (Fig. 7 A), consistent with previous observations in a similarly truncated Kir6.2 (Tucker et al. 1997; Drain et al. 1998; Trapp et al. 1998). Kir6.2ΔC35 currents also ran down after patch excision, albeit somewhat slower than when in the presence of SUR2. PPIs restored channel activity after run-down, providing further evidence that this effect is mediated through an interaction on Kir6.2 (Fig. 7B and Fig. C, and Table ). The increase in Po after reactivation by PPIs was reflected in the prolongation of mean open time and mean burst duration, and the profound shortening of mean closed time (Table ).


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)

PPIs reactivate KATP current in Kir6.2ΔC35. Single-channel currents were recorded from an inside-out patch from a COS-1 cell transfected with Kir6.2ΔC35. c– indicates the closed open level. The bottom trace in each panel represents a detail at a greater time resolution of the record in the top panel. The diagonal lines indicate the part of the top record that was selected for display in the lower record. (A) KATP current recorded immediately after excision of the patch before run-down. (B) Current recorded 10 min after excision of the patch after the current had run down. (C) Current recorded after a 5-min treatment with PPIs (1 mg/ml).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230641&req=5

Figure 7: PPIs reactivate KATP current in Kir6.2ΔC35. Single-channel currents were recorded from an inside-out patch from a COS-1 cell transfected with Kir6.2ΔC35. c– indicates the closed open level. The bottom trace in each panel represents a detail at a greater time resolution of the record in the top panel. The diagonal lines indicate the part of the top record that was selected for display in the lower record. (A) KATP current recorded immediately after excision of the patch before run-down. (B) Current recorded 10 min after excision of the patch after the current had run down. (C) Current recorded after a 5-min treatment with PPIs (1 mg/ml).
Mentions: Which subunit, SUR2 or Kir6.2, interacts with PPIs to cause the antagonism of ATP inhibition? Previously, using site-directed mutagenesis, we have shown that the reactivation effect of PPIs on KATP was through an interaction on the Kir6.2 subunit (Fan and Makielski 1997). Moreover, phosphatidylinositol is known to have a direct interaction with other inwardly rectifying K+ channels (Fan and Makielski 1997; Huang et al. 1998). Therefore, we explored whether the ATP desensitization effect of PPIs also localized to the channel subunit. When Kir6.2 was expressed alone, either with or without a green florescence protein tag on its COOH terminus (John et al. 1998), or with COOH-terminal truncation (Tucker et al. 1997), Kir6.2 retained ATP sensitivity, albeit reduced. We examined the effect of PPIs on ATP inhibition of the COOH-terminal truncated Kir6.2, Kir6.2ΔC35, that when expressed alone without SUR2 gave KATP currents. In the presence of low [ATP] (1 μM), the gating behavior of Kir6.2ΔC35 was characterized by short openings and less obvious short bursts (Fig. 7 A), consistent with previous observations in a similarly truncated Kir6.2 (Tucker et al. 1997; Drain et al. 1998; Trapp et al. 1998). Kir6.2ΔC35 currents also ran down after patch excision, albeit somewhat slower than when in the presence of SUR2. PPIs restored channel activity after run-down, providing further evidence that this effect is mediated through an interaction on Kir6.2 (Fig. 7B and Fig. C, and Table ). The increase in Po after reactivation by PPIs was reflected in the prolongation of mean open time and mean burst duration, and the profound shortening of mean closed time (Table ).

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