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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 abolishes high-affinity tolbutamide inhibition of Kir6.2+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 [tolbutamide], [ATP], or [Pip2] as indicated. The dashed line indicates zero current (determined in 5 mM ATP). (B) Percent tolbutamide inhibition versus time in Pip2 for four individual patches containing wild-type KATP channels. (C) Percent inhibition by ATP or tolbutamide before (Precontrol) or after (Post Pip2) application of Pip2 (2–4 min) from patches in B. (D) Plot of the change in percent tolbutamide inhibition versus change in K1/2,ATP after application of Pip2 for patches from B.
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Figure 2: Pip2 abolishes high-affinity tolbutamide inhibition of Kir6.2+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 [tolbutamide], [ATP], or [Pip2] as indicated. The dashed line indicates zero current (determined in 5 mM ATP). (B) Percent tolbutamide inhibition versus time in Pip2 for four individual patches containing wild-type KATP channels. (C) Percent inhibition by ATP or tolbutamide before (Precontrol) or after (Post Pip2) application of Pip2 (2–4 min) from patches in B. (D) Plot of the change in percent tolbutamide inhibition versus change in K1/2,ATP after application of Pip2 for patches from B.

Mentions: We can explore the correlation between tolbutamide sensitivity and open-state stability of the channel by applying PIP2. PIP2 increases the channel open probability by increasing bursting behavior of the single channel and decreases the sensitivity to ATP (Baukrowitz et al. 1998; Shyng and Nichols 1998). Although direct experimental proof is not available, both actions can be explained by models in which the action of PIP2 is to stabilize the channel open or bursting state, with ATP binding to, and stabilizing, the channel closed state (Shyng et al. 1997a). As shown in Fig. 2, treatment of wild-type Kir6.2+SUR1 channels with PIP2 leads to increased overall channel activity and loss of ATP sensitivity (Baukrowitz et al. 1998; Shyng and Nichols 1998). Concomitant with this increase in open-state stability, there is a gradual and complete loss of high affinity tolbutamide block (Fig. 2A and Fig. C). The rate of loss of both ATP sensitivity and high tolbutamide sensitivity (Fig. 2 B) are quite variable from patch to patch. However, there is a reasonable correlation between the tolbutamide inhibition and ATP sensitivity (Fig. 2 D).


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 abolishes high-affinity tolbutamide inhibition of Kir6.2+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 [tolbutamide], [ATP], or [Pip2] as indicated. The dashed line indicates zero current (determined in 5 mM ATP). (B) Percent tolbutamide inhibition versus time in Pip2 for four individual patches containing wild-type KATP channels. (C) Percent inhibition by ATP or tolbutamide before (Precontrol) or after (Post Pip2) application of Pip2 (2–4 min) from patches in B. (D) Plot of the change in percent tolbutamide inhibition versus change in K1/2,ATP after application of Pip2 for patches from B.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Pip2 abolishes high-affinity tolbutamide inhibition of Kir6.2+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 [tolbutamide], [ATP], or [Pip2] as indicated. The dashed line indicates zero current (determined in 5 mM ATP). (B) Percent tolbutamide inhibition versus time in Pip2 for four individual patches containing wild-type KATP channels. (C) Percent inhibition by ATP or tolbutamide before (Precontrol) or after (Post Pip2) application of Pip2 (2–4 min) from patches in B. (D) Plot of the change in percent tolbutamide inhibition versus change in K1/2,ATP after application of Pip2 for patches from B.
Mentions: We can explore the correlation between tolbutamide sensitivity and open-state stability of the channel by applying PIP2. PIP2 increases the channel open probability by increasing bursting behavior of the single channel and decreases the sensitivity to ATP (Baukrowitz et al. 1998; Shyng and Nichols 1998). Although direct experimental proof is not available, both actions can be explained by models in which the action of PIP2 is to stabilize the channel open or bursting state, with ATP binding to, and stabilizing, the channel closed state (Shyng et al. 1997a). As shown in Fig. 2, treatment of wild-type Kir6.2+SUR1 channels with PIP2 leads to increased overall channel activity and loss of ATP sensitivity (Baukrowitz et al. 1998; Shyng and Nichols 1998). Concomitant with this increase in open-state stability, there is a gradual and complete loss of high affinity tolbutamide block (Fig. 2A and Fig. C). The rate of loss of both ATP sensitivity and high tolbutamide sensitivity (Fig. 2 B) are quite variable from patch to patch. However, there is a reasonable correlation between the tolbutamide inhibition and ATP sensitivity (Fig. 2 D).

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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Related in: MedlinePlus