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Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels. Functional coupling of Kir6.2 and SUR1 subunits.

Koster JC, Sha Q, Nichols CG - J. Gen. Physiol. (1999)

Bottom Line: Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity.Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block.The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The sensitivity of K(ATP) channels to high-affinity block by sulfonylureas and to stimulation by K(+) channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP(2) drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant K(ATP) channels (Kir6. 2[DeltaN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP(2) also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP(2) application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP(2). The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.

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Pip2 effect on MgADP stimulation from cells expressing Kir6.2+SUR1 and Kir6.2[ΔN2-30]+SUR1 channels. (A) Representative currents from inside-out patches containing wild-type (Kir6.2+SUR1) or Kir6.2[ΔN2-30]+SUR1 channels. The patches were exposed to MgADP, MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (B) Plot of the percent MgADP stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent MgADP stimulation was determined by calculating the increase in current in the presence of MgADP and MgATP relative to current in MgATP alone, and then expressing this value as a fraction of the maximal current observed in the absence of ATP. (C) Percent stimulation by MgADP and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent MgADP stimulation versus K1/2,ATP after application of Pip2 to wild-type patches from B.
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Figure 5: Pip2 effect on MgADP stimulation from cells expressing Kir6.2+SUR1 and Kir6.2[ΔN2-30]+SUR1 channels. (A) Representative currents from inside-out patches containing wild-type (Kir6.2+SUR1) or Kir6.2[ΔN2-30]+SUR1 channels. The patches were exposed to MgADP, MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (B) Plot of the percent MgADP stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent MgADP stimulation was determined by calculating the increase in current in the presence of MgADP and MgATP relative to current in MgATP alone, and then expressing this value as a fraction of the maximal current observed in the absence of ATP. (C) Percent stimulation by MgADP and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent MgADP stimulation versus K1/2,ATP after application of Pip2 to wild-type patches from B.

Mentions: Activation of wild-type Kir6.2+SUR1 channels by MgADP and diazoxide, at a fixed [ATP], is quite variable from patch to patch (Fig. 5 B and 6 B). As shown in Fig. 5 A and 6 A, the ability of these agents to stimulate channel activity changes after PIP2 stimulation, and in a qualitatively similar way for both Kir6.2[ΔN2-30]+SUR1 and wild-type (Kir6.2+SUR1) channels. In each case, the stimulation tends to increase, but then gradually falls to zero with time after PIP2 application. The time course of this effect is also quite variable from patch to patch (Fig. 5 B and 6 B), but is reasonably well correlated with the accompanying change of ATP sensitivity (Fig. 5 D and 6 D). This result indicates that the stimulatory action of the PCOs, like ATP sensitivity itself, is not a fixed parameter of channel function, but is probably dependent on the open-state stability of the channel (Shyng and Nichols 1998). As PIP2 increasingly stabilizes the open state of the channel, sojourns in an ATP-accessible closed state become less and less frequent (Baukrowitz et al. 1998; Shyng and Nichols 1998). The present results are also consistent with PCOs acting by shifting the equilibrium between the open and closed states (see Shyng et al. 1997b), such that as the channel open-state stability approaches maximal, the stimulatory effect of the PCOs saturates.


Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels. Functional coupling of Kir6.2 and SUR1 subunits.

Koster JC, Sha Q, Nichols CG - J. Gen. Physiol. (1999)

Pip2 effect on MgADP stimulation from cells expressing Kir6.2+SUR1 and Kir6.2[ΔN2-30]+SUR1 channels. (A) Representative currents from inside-out patches containing wild-type (Kir6.2+SUR1) or Kir6.2[ΔN2-30]+SUR1 channels. The patches were exposed to MgADP, MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (B) Plot of the percent MgADP stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent MgADP stimulation was determined by calculating the increase in current in the presence of MgADP and MgATP relative to current in MgATP alone, and then expressing this value as a fraction of the maximal current observed in the absence of ATP. (C) Percent stimulation by MgADP and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent MgADP stimulation versus K1/2,ATP after application of Pip2 to wild-type patches from B.
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Figure 5: Pip2 effect on MgADP stimulation from cells expressing Kir6.2+SUR1 and Kir6.2[ΔN2-30]+SUR1 channels. (A) Representative currents from inside-out patches containing wild-type (Kir6.2+SUR1) or Kir6.2[ΔN2-30]+SUR1 channels. The patches were exposed to MgADP, MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (B) Plot of the percent MgADP stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent MgADP stimulation was determined by calculating the increase in current in the presence of MgADP and MgATP relative to current in MgATP alone, and then expressing this value as a fraction of the maximal current observed in the absence of ATP. (C) Percent stimulation by MgADP and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent MgADP stimulation versus K1/2,ATP after application of Pip2 to wild-type patches from B.
Mentions: Activation of wild-type Kir6.2+SUR1 channels by MgADP and diazoxide, at a fixed [ATP], is quite variable from patch to patch (Fig. 5 B and 6 B). As shown in Fig. 5 A and 6 A, the ability of these agents to stimulate channel activity changes after PIP2 stimulation, and in a qualitatively similar way for both Kir6.2[ΔN2-30]+SUR1 and wild-type (Kir6.2+SUR1) channels. In each case, the stimulation tends to increase, but then gradually falls to zero with time after PIP2 application. The time course of this effect is also quite variable from patch to patch (Fig. 5 B and 6 B), but is reasonably well correlated with the accompanying change of ATP sensitivity (Fig. 5 D and 6 D). This result indicates that the stimulatory action of the PCOs, like ATP sensitivity itself, is not a fixed parameter of channel function, but is probably dependent on the open-state stability of the channel (Shyng and Nichols 1998). As PIP2 increasingly stabilizes the open state of the channel, sojourns in an ATP-accessible closed state become less and less frequent (Baukrowitz et al. 1998; Shyng and Nichols 1998). The present results are also consistent with PCOs acting by shifting the equilibrium between the open and closed states (see Shyng et al. 1997b), such that as the channel open-state stability approaches maximal, the stimulatory effect of the PCOs saturates.

Bottom Line: Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity.Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block.The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The sensitivity of K(ATP) channels to high-affinity block by sulfonylureas and to stimulation by K(+) channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP(2) drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant K(ATP) channels (Kir6. 2[DeltaN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP(2) also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP(2) application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP(2). The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.

Show MeSH
Related in: MedlinePlus