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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 diazoxide 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 diazoxide and MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (Top) Open probability, estimated from noise analysis of 10 s of current in zero ATP, is indicated. (B) Plot of the percent diazoxide stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent diazoxide stimulation was determined by calculating the increase in current in the presence of diazoxide 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 diazoxide and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent diazoxide stimulation versus K1/2,ATP after application of Pip2 to patches from B.
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Figure 6: Pip2 effect on diazoxide 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 diazoxide and MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (Top) Open probability, estimated from noise analysis of 10 s of current in zero ATP, is indicated. (B) Plot of the percent diazoxide stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent diazoxide stimulation was determined by calculating the increase in current in the presence of diazoxide 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 diazoxide and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent diazoxide stimulation versus K1/2,ATP after application of Pip2 to patches from B.

Mentions: It is clear that PIP2 activation of KATP channels and other inward rectifiers does not require the presence of a SUR1 subunit, and probably results from a direct interaction of PIP2 with the cytoplasmic portion of the channel protein itself (Hilgemann and Ball 1996; Fan and Makielski 1997; Baukrowitz et al. 1998; Huang et al. 1998; Shyng and Nichols 1998). The present results indicate that PCO sensitivity, like ATP sensitivity (Baukrowitz et al. 1998; Shyng and Nichols 1998) is a variable, dynamically dependent on membrane phospholipid levels rather than a fixed parameter. After PIP2 application to inside-out patches, there is generally first an increase in the stimulatory action of PCOs, and then a gradual disappearance of their action as the PIP2 stimulation saturates, such that, even though ATP inhibition is still observable at high concentrations, there is no relief of this inhibition by PCOs (Fig. 5 and Fig. 6). As discussed in Shyng and Nichols 1998 and Baukrowitz et al. 1998, it is likely that membrane phospholipid levels are variable from cell to cell, and that such variability accounts for the cell-to-cell variability of ATP sensitivity that is observed physiologically (Findlay and Faivre 1991). By the same reasoning, the variable stimulatory action of PCOs (see, e.g., Fig. 5 B and 6 B) might be a result of cell-to-cell variability of membrane phospholipid levels. The results raise the question: How does the membrane phospholipid level determine the PCO sensitivity? One possibility is that PIP2 affects ATP hydrolysis, or PCO binding to the SUR1 subunit. However, as we have previously suggested (Shyng et al. 1997b), it seems likely that PCOs act ultimately to stabilize the open state of the channel itself, just as the phospholipids do. Therefore, the lack of PCO effects after elevation of phospholipids, is likely to be a consequence of the convergent action of these two agents on the energetic stability of the open state relative to the ATP-accessible closed state.


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 diazoxide 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 diazoxide and MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (Top) Open probability, estimated from noise analysis of 10 s of current in zero ATP, is indicated. (B) Plot of the percent diazoxide stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent diazoxide stimulation was determined by calculating the increase in current in the presence of diazoxide 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 diazoxide and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent diazoxide stimulation versus K1/2,ATP after application of Pip2 to patches from B.
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Figure 6: Pip2 effect on diazoxide 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 diazoxide and MgATP, or Pip2 as indicated. The dashed line represents zero current determined in 5 mM ATP. (Top) Open probability, estimated from noise analysis of 10 s of current in zero ATP, is indicated. (B) Plot of the percent diazoxide stimulation versus time in Pip2 for five individual patches containing wild-type KATP channels. Percent diazoxide stimulation was determined by calculating the increase in current in the presence of diazoxide 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 diazoxide and inhibition by ATP before (Precontrol) or after (Post Pip2) application of Pip2 (3–20 min) from patches in B. (D) Percent diazoxide stimulation versus K1/2,ATP after application of Pip2 to patches from B.
Mentions: It is clear that PIP2 activation of KATP channels and other inward rectifiers does not require the presence of a SUR1 subunit, and probably results from a direct interaction of PIP2 with the cytoplasmic portion of the channel protein itself (Hilgemann and Ball 1996; Fan and Makielski 1997; Baukrowitz et al. 1998; Huang et al. 1998; Shyng and Nichols 1998). The present results indicate that PCO sensitivity, like ATP sensitivity (Baukrowitz et al. 1998; Shyng and Nichols 1998) is a variable, dynamically dependent on membrane phospholipid levels rather than a fixed parameter. After PIP2 application to inside-out patches, there is generally first an increase in the stimulatory action of PCOs, and then a gradual disappearance of their action as the PIP2 stimulation saturates, such that, even though ATP inhibition is still observable at high concentrations, there is no relief of this inhibition by PCOs (Fig. 5 and Fig. 6). As discussed in Shyng and Nichols 1998 and Baukrowitz et al. 1998, it is likely that membrane phospholipid levels are variable from cell to cell, and that such variability accounts for the cell-to-cell variability of ATP sensitivity that is observed physiologically (Findlay and Faivre 1991). By the same reasoning, the variable stimulatory action of PCOs (see, e.g., Fig. 5 B and 6 B) might be a result of cell-to-cell variability of membrane phospholipid levels. The results raise the question: How does the membrane phospholipid level determine the PCO sensitivity? One possibility is that PIP2 affects ATP hydrolysis, or PCO binding to the SUR1 subunit. However, as we have previously suggested (Shyng et al. 1997b), it seems likely that PCOs act ultimately to stabilize the open state of the channel itself, just as the phospholipids do. Therefore, the lack of PCO effects after elevation of phospholipids, is likely to be a consequence of the convergent action of these two agents on the energetic stability of the open state relative to the ATP-accessible closed state.

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