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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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Mechanism of the PIP2-induced loss of tolbutamide block. (A) Representative current recorded from inside-out membrane patches containing wild-type KATP channels exposed to differing [tolbutamide] or 30 μM ATP, as shown. The gaps in the record are 4.5, 2, and 1 min. The dashed line indicates zero current (determined in 5 mM ATP). (B) Plot of relative current (Irel) versus [tolbutamide] for trace segments shown in A, both before (•) and with time after (○) Pip2 application. Data at 10 mM tolbutamide are from control and 10-min time points. Fitted lines correspond to a least squares fit of the sum of two Hill equations as described in Fig. 1with the fraction of high affinity inhibition allowed to vary (37, 23, 13, 4, 0% at 0, 4.5, 7, 8 and 10 min) as indicated, after PIP2 application. The insert shows data only at 7 min. The dashed lines correspond to (A, dashed) the same curve as above, and (B, dotted) a least squares fit of the sum of two Hill equations, with the fraction of high affinity inhibition held constant at 37%, but with the affinity (i.e., K1/2) allowed to vary. It is clear that high affinity block disappears because the high affinity fraction disappears, not because there is a change in the sensitivity of this component.
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Figure 3: Mechanism of the PIP2-induced loss of tolbutamide block. (A) Representative current recorded from inside-out membrane patches containing wild-type KATP channels exposed to differing [tolbutamide] or 30 μM ATP, as shown. The gaps in the record are 4.5, 2, and 1 min. The dashed line indicates zero current (determined in 5 mM ATP). (B) Plot of relative current (Irel) versus [tolbutamide] for trace segments shown in A, both before (•) and with time after (○) Pip2 application. Data at 10 mM tolbutamide are from control and 10-min time points. Fitted lines correspond to a least squares fit of the sum of two Hill equations as described in Fig. 1with the fraction of high affinity inhibition allowed to vary (37, 23, 13, 4, 0% at 0, 4.5, 7, 8 and 10 min) as indicated, after PIP2 application. The insert shows data only at 7 min. The dashed lines correspond to (A, dashed) the same curve as above, and (B, dotted) a least squares fit of the sum of two Hill equations, with the fraction of high affinity inhibition held constant at 37%, but with the affinity (i.e., K1/2) allowed to vary. It is clear that high affinity block disappears because the high affinity fraction disappears, not because there is a change in the sensitivity of this component.

Mentions: The loss of high affinity tolbutamide inhibition could occur because the high affinity component actually changes affinity (i.e., the real, or apparent, binding affinity of tolbutamide is reduced), or because high affinity binding fails to cause inhibition of channel activity. As shown in Fig. 3, the latter explanation is correct; with time after addition of PIP2, the dose–response relationship can be fit by assuming that the high affinity inhibition becomes a progressively smaller fraction of the [tolbutamide]-inhibition relationship. Data points at intermediate times cannot be fit by assuming a constant high affinity fraction, with reduced affinity. This is consistent with an effect of PIP2 on the coupling of high affinity binding to channel inhibition, not on modifying tolbutamide binding itself.


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)

Mechanism of the PIP2-induced loss of tolbutamide block. (A) Representative current recorded from inside-out membrane patches containing wild-type KATP channels exposed to differing [tolbutamide] or 30 μM ATP, as shown. The gaps in the record are 4.5, 2, and 1 min. The dashed line indicates zero current (determined in 5 mM ATP). (B) Plot of relative current (Irel) versus [tolbutamide] for trace segments shown in A, both before (•) and with time after (○) Pip2 application. Data at 10 mM tolbutamide are from control and 10-min time points. Fitted lines correspond to a least squares fit of the sum of two Hill equations as described in Fig. 1with the fraction of high affinity inhibition allowed to vary (37, 23, 13, 4, 0% at 0, 4.5, 7, 8 and 10 min) as indicated, after PIP2 application. The insert shows data only at 7 min. The dashed lines correspond to (A, dashed) the same curve as above, and (B, dotted) a least squares fit of the sum of two Hill equations, with the fraction of high affinity inhibition held constant at 37%, but with the affinity (i.e., K1/2) allowed to vary. It is clear that high affinity block disappears because the high affinity fraction disappears, not because there is a change in the sensitivity of this component.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230640&req=5

Figure 3: Mechanism of the PIP2-induced loss of tolbutamide block. (A) Representative current recorded from inside-out membrane patches containing wild-type KATP channels exposed to differing [tolbutamide] or 30 μM ATP, as shown. The gaps in the record are 4.5, 2, and 1 min. The dashed line indicates zero current (determined in 5 mM ATP). (B) Plot of relative current (Irel) versus [tolbutamide] for trace segments shown in A, both before (•) and with time after (○) Pip2 application. Data at 10 mM tolbutamide are from control and 10-min time points. Fitted lines correspond to a least squares fit of the sum of two Hill equations as described in Fig. 1with the fraction of high affinity inhibition allowed to vary (37, 23, 13, 4, 0% at 0, 4.5, 7, 8 and 10 min) as indicated, after PIP2 application. The insert shows data only at 7 min. The dashed lines correspond to (A, dashed) the same curve as above, and (B, dotted) a least squares fit of the sum of two Hill equations, with the fraction of high affinity inhibition held constant at 37%, but with the affinity (i.e., K1/2) allowed to vary. It is clear that high affinity block disappears because the high affinity fraction disappears, not because there is a change in the sensitivity of this component.
Mentions: The loss of high affinity tolbutamide inhibition could occur because the high affinity component actually changes affinity (i.e., the real, or apparent, binding affinity of tolbutamide is reduced), or because high affinity binding fails to cause inhibition of channel activity. As shown in Fig. 3, the latter explanation is correct; with time after addition of PIP2, the dose–response relationship can be fit by assuming that the high affinity inhibition becomes a progressively smaller fraction of the [tolbutamide]-inhibition relationship. Data points at intermediate times cannot be fit by assuming a constant high affinity fraction, with reduced affinity. This is consistent with an effect of PIP2 on the coupling of high affinity binding to channel inhibition, not on modifying tolbutamide binding itself.

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