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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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ATP block of Kir6.2Δ35 before and after treatment with PPIs (1 mg/ml). (A) KATP currents from single-channels recorded at the [ATP] indicated. Traces at left were obtained before treatment with PPIs; at right traces were obtained 10 min after treatment. c– and o– indicate closed and open current levels, respectively. (B) Concentration–response relationship of ATP block before and after treatment of Kir6.2ΔC35 with PPIs. Symbols and error bars represent the mean ± SE (n = 1–4 for each data point) in control and 10 min after treatment. Lines represent fitting of the data to a binding equation as in Fig. 2. Ki is the concentration of half inhibition and S is the slope factor or Hill coefficients. Ki = 247 ± 188 μm in control, and 2.17 ± 0.93 mM after treatment with PPIs (P < 0.001). S = 1.3 and 1.4 before and after treatment with PPIs, respectively.
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Figure 8: ATP block of Kir6.2Δ35 before and after treatment with PPIs (1 mg/ml). (A) KATP currents from single-channels recorded at the [ATP] indicated. Traces at left were obtained before treatment with PPIs; at right traces were obtained 10 min after treatment. c– and o– indicate closed and open current levels, respectively. (B) Concentration–response relationship of ATP block before and after treatment of Kir6.2ΔC35 with PPIs. Symbols and error bars represent the mean ± SE (n = 1–4 for each data point) in control and 10 min after treatment. Lines represent fitting of the data to a binding equation as in Fig. 2. Ki is the concentration of half inhibition and S is the slope factor or Hill coefficients. Ki = 247 ± 188 μm in control, and 2.17 ± 0.93 mM after treatment with PPIs (P < 0.001). S = 1.3 and 1.4 before and after treatment with PPIs, respectively.

Mentions: Representative traces from a patch with a single Kir6.2ΔC35 channel (Fig. 8 A) show that its kinetics are distinctly different from those of native KATP or cloned SUR2/Kir6.2 KATP currents. They also show that Kir6.2ΔC35 retains some sensitivity to ATP inhibition in control and that PPIs also desensitize ATP inhibition for this channel. Summary data for the ATP inhibition of Kir6.2ΔC35 (Fig. 8 B) show that Ki in the control was 247 μM for ATP inhibition, compared with 35 μM for SUR2/Kir6.2 (Fig. 2), a sevenfold difference. Thus, the truncated version of the channel is much less sensitive than in the presence of the SUR subunit. These features of COOH-terminal truncated Kir6.2 are consistent with the data published by Tucker et al. 1997 for the truncated Kir6.2 and those by John et al. 1998 for the full length of Kir6.2. As with SUR2/Kir6.2, treatment with PPIs (10 min, 1 mg/ml) significantly (P < 0.001) shifted the concentration–response curve for ATP inhibition to the right, resulting in a larger value of Ki of 2.1 mM (Fig. 8 D). However, in SUR2/Kir6.2 the same treatment desensitized Ki by nearly 500-fold from 35 μM to 16 mM (Fig. 2). These results suggest that although the essential effect of PPIs on reactivation and desensitization of ATP inhibition are retained in Kir6.2ΔC35, differences do exist. The SUR subunit and/or the alteration to the COOH terminus may modulate the effects of PPIs.


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)

ATP block of Kir6.2Δ35 before and after treatment with PPIs (1 mg/ml). (A) KATP currents from single-channels recorded at the [ATP] indicated. Traces at left were obtained before treatment with PPIs; at right traces were obtained 10 min after treatment. c– and o– indicate closed and open current levels, respectively. (B) Concentration–response relationship of ATP block before and after treatment of Kir6.2ΔC35 with PPIs. Symbols and error bars represent the mean ± SE (n = 1–4 for each data point) in control and 10 min after treatment. Lines represent fitting of the data to a binding equation as in Fig. 2. Ki is the concentration of half inhibition and S is the slope factor or Hill coefficients. Ki = 247 ± 188 μm in control, and 2.17 ± 0.93 mM after treatment with PPIs (P < 0.001). S = 1.3 and 1.4 before and after treatment with PPIs, respectively.
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Figure 8: ATP block of Kir6.2Δ35 before and after treatment with PPIs (1 mg/ml). (A) KATP currents from single-channels recorded at the [ATP] indicated. Traces at left were obtained before treatment with PPIs; at right traces were obtained 10 min after treatment. c– and o– indicate closed and open current levels, respectively. (B) Concentration–response relationship of ATP block before and after treatment of Kir6.2ΔC35 with PPIs. Symbols and error bars represent the mean ± SE (n = 1–4 for each data point) in control and 10 min after treatment. Lines represent fitting of the data to a binding equation as in Fig. 2. Ki is the concentration of half inhibition and S is the slope factor or Hill coefficients. Ki = 247 ± 188 μm in control, and 2.17 ± 0.93 mM after treatment with PPIs (P < 0.001). S = 1.3 and 1.4 before and after treatment with PPIs, respectively.
Mentions: Representative traces from a patch with a single Kir6.2ΔC35 channel (Fig. 8 A) show that its kinetics are distinctly different from those of native KATP or cloned SUR2/Kir6.2 KATP currents. They also show that Kir6.2ΔC35 retains some sensitivity to ATP inhibition in control and that PPIs also desensitize ATP inhibition for this channel. Summary data for the ATP inhibition of Kir6.2ΔC35 (Fig. 8 B) show that Ki in the control was 247 μM for ATP inhibition, compared with 35 μM for SUR2/Kir6.2 (Fig. 2), a sevenfold difference. Thus, the truncated version of the channel is much less sensitive than in the presence of the SUR subunit. These features of COOH-terminal truncated Kir6.2 are consistent with the data published by Tucker et al. 1997 for the truncated Kir6.2 and those by John et al. 1998 for the full length of Kir6.2. As with SUR2/Kir6.2, treatment with PPIs (10 min, 1 mg/ml) significantly (P < 0.001) shifted the concentration–response curve for ATP inhibition to the right, resulting in a larger value of Ki of 2.1 mM (Fig. 8 D). However, in SUR2/Kir6.2 the same treatment desensitized Ki by nearly 500-fold from 35 μM to 16 mM (Fig. 2). These results suggest that although the essential effect of PPIs on reactivation and desensitization of ATP inhibition are retained in Kir6.2ΔC35, differences do exist. The SUR subunit and/or the alteration to the COOH terminus may modulate the effects of PPIs.

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