Limits...
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.

Show MeSH

Related in: MedlinePlus

Concentration-response for ATP inhibition of KATP. Ascending and descending [ATP] response relationships in a single multichannel patch before (A) and after (B) treatment with PPIs (10 min, 1 mg/ml). KATP currents were recorded in inside-out patches from COS-1 cells transfected with SUR2/Kir6.2. Bars and numbers represent ATP concentration except that in B 10K stands for an internal solution with 10 mM [K+] that produced a 0 current level for equimolar [K+] at 0 mV. (C) Summary data for the ATP block concentration–response relationship before and after treatment with PPIs from experiments such as those shown in A and B. Symbols and error bars represent the mean ± SE from four control experiments and three or four experiments after treatment. Summary data for ATP sensitivity was obtained by fitting the dependence of normalized Po on [ATP] using the expression: P = Po,max {1 − 1 /[1 + (Ki/[ATP])S]} where P is the normalized Po, in ATP relative to the maximal Po (Po,max) in the absence of ATP or to 1 μM [ATP] that produced little or no inhibition; Ki is the half-inhibitory [ATP]; and S is the slope-factor or Hill coefficient. Po,max, Ki, and S were free parameters for fitting. Before treatment with PPIs, Ki = 34.9 ± 6.7 μM, and after treatment Ki = 15.6 ± 2.7 mM. (P < 0.001). The Hill coefficients S were 1.03 ± 0.11 versus 0.94 ± 0.17, respectively. In the inset, PPIs stands for the data collected after the patches was treated for 10 min with PPIs (1 mg/ml) and PPIs were washed out. The same label is used in the subsequent figure legends.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230641&req=5

Figure 2: Concentration-response for ATP inhibition of KATP. Ascending and descending [ATP] response relationships in a single multichannel patch before (A) and after (B) treatment with PPIs (10 min, 1 mg/ml). KATP currents were recorded in inside-out patches from COS-1 cells transfected with SUR2/Kir6.2. Bars and numbers represent ATP concentration except that in B 10K stands for an internal solution with 10 mM [K+] that produced a 0 current level for equimolar [K+] at 0 mV. (C) Summary data for the ATP block concentration–response relationship before and after treatment with PPIs from experiments such as those shown in A and B. Symbols and error bars represent the mean ± SE from four control experiments and three or four experiments after treatment. Summary data for ATP sensitivity was obtained by fitting the dependence of normalized Po on [ATP] using the expression: P = Po,max {1 − 1 /[1 + (Ki/[ATP])S]} where P is the normalized Po, in ATP relative to the maximal Po (Po,max) in the absence of ATP or to 1 μM [ATP] that produced little or no inhibition; Ki is the half-inhibitory [ATP]; and S is the slope-factor or Hill coefficient. Po,max, Ki, and S were free parameters for fitting. Before treatment with PPIs, Ki = 34.9 ± 6.7 μM, and after treatment Ki = 15.6 ± 2.7 mM. (P < 0.001). The Hill coefficients S were 1.03 ± 0.11 versus 0.94 ± 0.17, respectively. In the inset, PPIs stands for the data collected after the patches was treated for 10 min with PPIs (1 mg/ml) and PPIs were washed out. The same label is used in the subsequent figure legends.

Mentions: With longer (>3 min) exposure, PPIs dramatically desensitized KATP to ATP inhibition. When PPIs were applied in the continuous presence of 1 mM ATP (Fig. 1 B), little or no KATP activity was observed within the initial 30 s, demonstrating maintained ATP inhibition at a time when the effect of PPIs on maximal Po was nearly complete. However, after several minutes in PPIs, KATP activity gradually increased in the presence of 1 mM ATP, demonstrating a loss of ATP sensitivity, and activity reached a new stable level after ∼10 min. Similar results were obtained in five patches: one from a dog ventricular cell, three from rat ventricular cells, and one from SUR2/Kir6.2. In the particular experiment shown in Fig. 1 B, KATP had already partially run down before application of PPIs. However, loss of ATP sensitivity with PPIs required neither previous channel run-down nor ATP, nor did it require the continuous presence of PPIs in the bathing solution as was demonstrated in experiments similar to that shown in Fig. 1 C. In a patch (SUR2/Kir6.2) without notable prior run-down (as defined by the ratio noted above), ATP sensitivity was first demonstrated by exposure to 1 mM ATP, then PPIs were applied for 10 min in the absence of inhibitory [ATP]. Desensitization of ATP inhibition was then demonstrated by the failure of 1 mM ATP to suppress KATP currents after removal of the PPIs from the bath solution (Fig. 1 C). In these experiments, slight recovery of ATP sensitivity was observed 7–20 min after PPIs were removed (data not shown). Interestingly, in those patches treated with PPIs where KATP activity was followed for several hours, ATP sensitivity never returned to its initial value, while at the same time the maximal Po gradually decreased to nearly zero. The concentration–response of ATP inhibition before and after exposure to PPIs for 10 min was measured within single patches (SUR2/Kir6.2). [ATP] was stepped between 1 and 1,000 μM in control and between 1 and 10,000 μM after a 10-min exposure to PPIs (for example, see Fig. 2). [ATP] changes in 10-s intervals were stepped in both increasing and decreasing concentrations. As studied in four patches (Fig. 2 C), PPIs desensitized ATP sensitivity by increasing the Ki for inhibition nearly 500-fold (before PPIs: Ki = 34.9 ± 6.7 μM; after PPIs: Ki = 15.6 ± 2.7 mM, P < 0.001) without a significant difference in slope factor (1.03 ± 0.11 versus 0.94 ± 0.17, before versus after PPIs, respectively). For 1 mg/ml phosphatidylcholine, an uncharged phospholipid, a small, variable, statistically insignificant effect (n = 7) was observed on the KATP sensitivity of native rat cardiac myocytes. This result suggested a critical role of the negatively charged head group for the effect on ATP sensitivity. Previously, we had found that negatively charged groups of phosphatidylinositol were needed for reactivation of KATP (Fan and Makielski 1997).


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)

Concentration-response for ATP inhibition of KATP. Ascending and descending [ATP] response relationships in a single multichannel patch before (A) and after (B) treatment with PPIs (10 min, 1 mg/ml). KATP currents were recorded in inside-out patches from COS-1 cells transfected with SUR2/Kir6.2. Bars and numbers represent ATP concentration except that in B 10K stands for an internal solution with 10 mM [K+] that produced a 0 current level for equimolar [K+] at 0 mV. (C) Summary data for the ATP block concentration–response relationship before and after treatment with PPIs from experiments such as those shown in A and B. Symbols and error bars represent the mean ± SE from four control experiments and three or four experiments after treatment. Summary data for ATP sensitivity was obtained by fitting the dependence of normalized Po on [ATP] using the expression: P = Po,max {1 − 1 /[1 + (Ki/[ATP])S]} where P is the normalized Po, in ATP relative to the maximal Po (Po,max) in the absence of ATP or to 1 μM [ATP] that produced little or no inhibition; Ki is the half-inhibitory [ATP]; and S is the slope-factor or Hill coefficient. Po,max, Ki, and S were free parameters for fitting. Before treatment with PPIs, Ki = 34.9 ± 6.7 μM, and after treatment Ki = 15.6 ± 2.7 mM. (P < 0.001). The Hill coefficients S were 1.03 ± 0.11 versus 0.94 ± 0.17, respectively. In the inset, PPIs stands for the data collected after the patches was treated for 10 min with PPIs (1 mg/ml) and PPIs were washed out. The same label is used in the subsequent figure legends.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Concentration-response for ATP inhibition of KATP. Ascending and descending [ATP] response relationships in a single multichannel patch before (A) and after (B) treatment with PPIs (10 min, 1 mg/ml). KATP currents were recorded in inside-out patches from COS-1 cells transfected with SUR2/Kir6.2. Bars and numbers represent ATP concentration except that in B 10K stands for an internal solution with 10 mM [K+] that produced a 0 current level for equimolar [K+] at 0 mV. (C) Summary data for the ATP block concentration–response relationship before and after treatment with PPIs from experiments such as those shown in A and B. Symbols and error bars represent the mean ± SE from four control experiments and three or four experiments after treatment. Summary data for ATP sensitivity was obtained by fitting the dependence of normalized Po on [ATP] using the expression: P = Po,max {1 − 1 /[1 + (Ki/[ATP])S]} where P is the normalized Po, in ATP relative to the maximal Po (Po,max) in the absence of ATP or to 1 μM [ATP] that produced little or no inhibition; Ki is the half-inhibitory [ATP]; and S is the slope-factor or Hill coefficient. Po,max, Ki, and S were free parameters for fitting. Before treatment with PPIs, Ki = 34.9 ± 6.7 μM, and after treatment Ki = 15.6 ± 2.7 mM. (P < 0.001). The Hill coefficients S were 1.03 ± 0.11 versus 0.94 ± 0.17, respectively. In the inset, PPIs stands for the data collected after the patches was treated for 10 min with PPIs (1 mg/ml) and PPIs were washed out. The same label is used in the subsequent figure legends.
Mentions: With longer (>3 min) exposure, PPIs dramatically desensitized KATP to ATP inhibition. When PPIs were applied in the continuous presence of 1 mM ATP (Fig. 1 B), little or no KATP activity was observed within the initial 30 s, demonstrating maintained ATP inhibition at a time when the effect of PPIs on maximal Po was nearly complete. However, after several minutes in PPIs, KATP activity gradually increased in the presence of 1 mM ATP, demonstrating a loss of ATP sensitivity, and activity reached a new stable level after ∼10 min. Similar results were obtained in five patches: one from a dog ventricular cell, three from rat ventricular cells, and one from SUR2/Kir6.2. In the particular experiment shown in Fig. 1 B, KATP had already partially run down before application of PPIs. However, loss of ATP sensitivity with PPIs required neither previous channel run-down nor ATP, nor did it require the continuous presence of PPIs in the bathing solution as was demonstrated in experiments similar to that shown in Fig. 1 C. In a patch (SUR2/Kir6.2) without notable prior run-down (as defined by the ratio noted above), ATP sensitivity was first demonstrated by exposure to 1 mM ATP, then PPIs were applied for 10 min in the absence of inhibitory [ATP]. Desensitization of ATP inhibition was then demonstrated by the failure of 1 mM ATP to suppress KATP currents after removal of the PPIs from the bath solution (Fig. 1 C). In these experiments, slight recovery of ATP sensitivity was observed 7–20 min after PPIs were removed (data not shown). Interestingly, in those patches treated with PPIs where KATP activity was followed for several hours, ATP sensitivity never returned to its initial value, while at the same time the maximal Po gradually decreased to nearly zero. The concentration–response of ATP inhibition before and after exposure to PPIs for 10 min was measured within single patches (SUR2/Kir6.2). [ATP] was stepped between 1 and 1,000 μM in control and between 1 and 10,000 μM after a 10-min exposure to PPIs (for example, see Fig. 2). [ATP] changes in 10-s intervals were stepped in both increasing and decreasing concentrations. As studied in four patches (Fig. 2 C), PPIs desensitized ATP sensitivity by increasing the Ki for inhibition nearly 500-fold (before PPIs: Ki = 34.9 ± 6.7 μM; after PPIs: Ki = 15.6 ± 2.7 mM, P < 0.001) without a significant difference in slope factor (1.03 ± 0.11 versus 0.94 ± 0.17, before versus after PPIs, respectively). For 1 mg/ml phosphatidylcholine, an uncharged phospholipid, a small, variable, statistically insignificant effect (n = 7) was observed on the KATP sensitivity of native rat cardiac myocytes. This result suggested a critical role of the negatively charged head group for the effect on ATP sensitivity. Previously, we had found that negatively charged groups of phosphatidylinositol were needed for reactivation of KATP (Fan and Makielski 1997).

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