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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 tolbutamide and PCO sensitivity is partially reversed by application of poly-l-lysine. (A) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide] or ATP, as shown. The dashed line represents a 28-min gap in the trace, during which time Pip2 (5μg/ml) was applied. After Pip2 application, poly-l-lysine (10 μg/ml) was applied as indicated. ATP and tolbutamide sensitivity as a function of time is shown below. Sensitivity was assessed as the activity remaining in ATP or tolbutamide relative to maximal current in zero ATP, zero tolbutamide. (B) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to MgATP and diazoxide, as indicated. The dashed lines represent 18- and 13-min gaps in the trace, during which time Pip2 and poly-l-lysine, respectively, were applied.
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Figure 7: Pip2 effect on tolbutamide and PCO sensitivity is partially reversed by application of poly-l-lysine. (A) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide] or ATP, as shown. The dashed line represents a 28-min gap in the trace, during which time Pip2 (5μg/ml) was applied. After Pip2 application, poly-l-lysine (10 μg/ml) was applied as indicated. ATP and tolbutamide sensitivity as a function of time is shown below. Sensitivity was assessed as the activity remaining in ATP or tolbutamide relative to maximal current in zero ATP, zero tolbutamide. (B) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to MgATP and diazoxide, as indicated. The dashed lines represent 18- and 13-min gaps in the trace, during which time Pip2 and poly-l-lysine, respectively, were applied.

Mentions: Treatment with polycations can reverse the stimulatory actions of PIP2 on open probability and ATP sensitivity (Deutsch et al. 1994; Shyng and Nichols 1998), probably by shielding the negative charges introduced by PIP2. As shown in Fig. 7, some reversal of both tolbutamide insensitivity and loss of PCO action is observed when patches are subsequently treated with poly-l-lysine. However, some irreversible loss of high affinity tolbutamide sensitivity, as well as of diazoxide and MgADP stimulation, also occurs after prolonged PIP2 treatment, such that poly-l-lysine may only partially restore the SUR1 coupling (e.g., Fig. 7 A), even though ATP sensitivity can be restored to, or beyond, control levels (see discussion).


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 tolbutamide and PCO sensitivity is partially reversed by application of poly-l-lysine. (A) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide] or ATP, as shown. The dashed line represents a 28-min gap in the trace, during which time Pip2 (5μg/ml) was applied. After Pip2 application, poly-l-lysine (10 μg/ml) was applied as indicated. ATP and tolbutamide sensitivity as a function of time is shown below. Sensitivity was assessed as the activity remaining in ATP or tolbutamide relative to maximal current in zero ATP, zero tolbutamide. (B) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to MgATP and diazoxide, as indicated. The dashed lines represent 18- and 13-min gaps in the trace, during which time Pip2 and poly-l-lysine, respectively, were applied.
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Figure 7: Pip2 effect on tolbutamide and PCO sensitivity is partially reversed by application of poly-l-lysine. (A) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide] or ATP, as shown. The dashed line represents a 28-min gap in the trace, during which time Pip2 (5μg/ml) was applied. After Pip2 application, poly-l-lysine (10 μg/ml) was applied as indicated. ATP and tolbutamide sensitivity as a function of time is shown below. Sensitivity was assessed as the activity remaining in ATP or tolbutamide relative to maximal current in zero ATP, zero tolbutamide. (B) Current recorded from representative inside-out membrane patch containing wild-type channels at −50 mV in Kint solution. The patch was exposed to MgATP and diazoxide, as indicated. The dashed lines represent 18- and 13-min gaps in the trace, during which time Pip2 and poly-l-lysine, respectively, were applied.
Mentions: Treatment with polycations can reverse the stimulatory actions of PIP2 on open probability and ATP sensitivity (Deutsch et al. 1994; Shyng and Nichols 1998), probably by shielding the negative charges introduced by PIP2. As shown in Fig. 7, some reversal of both tolbutamide insensitivity and loss of PCO action is observed when patches are subsequently treated with poly-l-lysine. However, some irreversible loss of high affinity tolbutamide sensitivity, as well as of diazoxide and MgADP stimulation, also occurs after prolonged PIP2 treatment, such that poly-l-lysine may only partially restore the SUR1 coupling (e.g., Fig. 7 A), even though ATP sensitivity can be restored to, or beyond, control levels (see discussion).

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