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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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Histograms of open and closed time distributions and kinetic properties of a single KATP channel recorded at 0 mV from a rat cardiac ventricular cell. (A) Closed time distribution analyzed from the same patch as shown in Fig. 4. The solid lines were fitted with a probability density function containing the sum of four exponential functions (). (B) Open time distribution. The solid lines were fitted with the sum of three exponential functions. For both A and B, the left panels are the control and the right panels are after 5-min treatment with PPIs (1 mg/ml). The parameters of probability density functions obtained from the fitting are listed in Table .
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Figure 5: Histograms of open and closed time distributions and kinetic properties of a single KATP channel recorded at 0 mV from a rat cardiac ventricular cell. (A) Closed time distribution analyzed from the same patch as shown in Fig. 4. The solid lines were fitted with a probability density function containing the sum of four exponential functions (). (B) Open time distribution. The solid lines were fitted with the sum of three exponential functions. For both A and B, the left panels are the control and the right panels are after 5-min treatment with PPIs (1 mg/ml). The parameters of probability density functions obtained from the fitting are listed in Table .

Mentions: Under these experimental conditions, untreated single-channel currents exhibited characteristic KATP kinetics (Fig. 4 A, Control) consistent with those reported in the literature (e.g., Davies et al. 1992). Current events were determined at > tmin = 150 μs resolution (see methods and example of idealized events in the top trace of Fig. 4 B). Current flickering was seen and resolved for all ATP concentrations in untreated and treated channels (Fig. 4 B). Also typical for this channel, the open channel activity occurred in bursts of activity separated by closed intervals longer than the critical time tcritical (see methods for determination of tcritical, which was generally 3–5 ms) and is best seen in Fig. 4 B. Even longer closures seen in the slower time scale (Fig. 4 A) isolated groups of bursts into clusters. Due to the difficulty in obtaining stable recordings of sufficient length, this cluster behavior was not further analyzed. A typical example of the channel activity histograms for a single-channel patch is shown in Fig. 5, and the parameters of the fits are given in 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)

Histograms of open and closed time distributions and kinetic properties of a single KATP channel recorded at 0 mV from a rat cardiac ventricular cell. (A) Closed time distribution analyzed from the same patch as shown in Fig. 4. The solid lines were fitted with a probability density function containing the sum of four exponential functions (). (B) Open time distribution. The solid lines were fitted with the sum of three exponential functions. For both A and B, the left panels are the control and the right panels are after 5-min treatment with PPIs (1 mg/ml). The parameters of probability density functions obtained from the fitting are listed in Table .
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Histograms of open and closed time distributions and kinetic properties of a single KATP channel recorded at 0 mV from a rat cardiac ventricular cell. (A) Closed time distribution analyzed from the same patch as shown in Fig. 4. The solid lines were fitted with a probability density function containing the sum of four exponential functions (). (B) Open time distribution. The solid lines were fitted with the sum of three exponential functions. For both A and B, the left panels are the control and the right panels are after 5-min treatment with PPIs (1 mg/ml). The parameters of probability density functions obtained from the fitting are listed in Table .
Mentions: Under these experimental conditions, untreated single-channel currents exhibited characteristic KATP kinetics (Fig. 4 A, Control) consistent with those reported in the literature (e.g., Davies et al. 1992). Current events were determined at > tmin = 150 μs resolution (see methods and example of idealized events in the top trace of Fig. 4 B). Current flickering was seen and resolved for all ATP concentrations in untreated and treated channels (Fig. 4 B). Also typical for this channel, the open channel activity occurred in bursts of activity separated by closed intervals longer than the critical time tcritical (see methods for determination of tcritical, which was generally 3–5 ms) and is best seen in Fig. 4 B. Even longer closures seen in the slower time scale (Fig. 4 A) isolated groups of bursts into clusters. Due to the difficulty in obtaining stable recordings of sufficient length, this cluster behavior was not further analyzed. A typical example of the channel activity histograms for a single-channel patch is shown in Fig. 5, and the parameters of the fits are given in 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