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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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High affinity tolbutamide sensitivity depends on open-state stability. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution. Patches were exposed to differing [tolbutamide] or 5 mM ATP, as shown. (B) Current recorded from representative patch containing Kir6.2 [R176A]+SUR1 channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide], ATP, or 5 μg/ml PIP2, as shown. The dashed lines represent 12- and 2-min gaps.
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Figure 4: High affinity tolbutamide sensitivity depends on open-state stability. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution. Patches were exposed to differing [tolbutamide] or 5 mM ATP, as shown. (B) Current recorded from representative patch containing Kir6.2 [R176A]+SUR1 channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide], ATP, or 5 μg/ml PIP2, as shown. The dashed lines represent 12- and 2-min gaps.

Mentions: Since PIP2 and NH2-terminal deletion both increase the channel open state stability (Fan and Makielski 1997; Shyng and Nichols 1998; Koster et al. 1999), the loss of high affinity tolbutamide sensitivity in NH2-terminal truncated channels, and on wild-type channels treated with PIP2 suggests that the coupling of high affinity tolbutamide binding (at SUR1; Aguilar-Bryan et al. 1995) to channel inhibition may also depend on the open-state stability. To examine this possibility further, we have measured the tolbutamide sensitivity of channels expressed from Kir6.2[R176A]+SUR1, and Kir6.2[L164A+SUR1] channels, which have intrinsically very low, and high, open-state stabilities, respectively (Shyng and Nichols 1998). Kir6.2[R176A]+SUR1 channels have a much lower intrinsic open probability in the absence of ATP (Pozero < 0.1) than wild-type channels (Pozero ∼ 0.5), due to reduced PIP2 affinity (Fan and Makielski 1997; Huang et al. 1998; Shyng and Nichols 1998). As shown in Fig. 4 A, it is clear that these channels have a larger high affinity component of tolbutamide inhibition than wild-type channels, but which is again lost as Pozero increases after treatment with PIP2 (Fig. 4 B). In contrast, Kir6.2[L164A]+SUR1 channels have a very high Pozero (>0.85), corresponding to an intrinsic ATP sensitivity of K1/2,ATP ∼ 1mM (data not shown), due to the open-state stabilizing effect of this mutation. As shown in Fig. 4 A, there is essentially no high affinity component of tolbutamide inhibition of Kir6.2[L164A]+SUR1 channels. On average, 100 μM tolbutamide inhibited wild-type Kir6.2+SUR1, Kir6.2[L164A]+SUR1, and Kir6.2[R176A]+SUR1 channels by 33 ± 3, 3 ± 2, and 77 ± 6%, respectively (n = 3 in each case).


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)

High affinity tolbutamide sensitivity depends on open-state stability. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution. Patches were exposed to differing [tolbutamide] or 5 mM ATP, as shown. (B) Current recorded from representative patch containing Kir6.2 [R176A]+SUR1 channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide], ATP, or 5 μg/ml PIP2, as shown. The dashed lines represent 12- and 2-min gaps.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: High affinity tolbutamide sensitivity depends on open-state stability. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution. Patches were exposed to differing [tolbutamide] or 5 mM ATP, as shown. (B) Current recorded from representative patch containing Kir6.2 [R176A]+SUR1 channels at −50 mV in Kint solution. The patch was exposed to differing [tolbutamide], ATP, or 5 μg/ml PIP2, as shown. The dashed lines represent 12- and 2-min gaps.
Mentions: Since PIP2 and NH2-terminal deletion both increase the channel open state stability (Fan and Makielski 1997; Shyng and Nichols 1998; Koster et al. 1999), the loss of high affinity tolbutamide sensitivity in NH2-terminal truncated channels, and on wild-type channels treated with PIP2 suggests that the coupling of high affinity tolbutamide binding (at SUR1; Aguilar-Bryan et al. 1995) to channel inhibition may also depend on the open-state stability. To examine this possibility further, we have measured the tolbutamide sensitivity of channels expressed from Kir6.2[R176A]+SUR1, and Kir6.2[L164A+SUR1] channels, which have intrinsically very low, and high, open-state stabilities, respectively (Shyng and Nichols 1998). Kir6.2[R176A]+SUR1 channels have a much lower intrinsic open probability in the absence of ATP (Pozero < 0.1) than wild-type channels (Pozero ∼ 0.5), due to reduced PIP2 affinity (Fan and Makielski 1997; Huang et al. 1998; Shyng and Nichols 1998). As shown in Fig. 4 A, it is clear that these channels have a larger high affinity component of tolbutamide inhibition than wild-type channels, but which is again lost as Pozero increases after treatment with PIP2 (Fig. 4 B). In contrast, Kir6.2[L164A]+SUR1 channels have a very high Pozero (>0.85), corresponding to an intrinsic ATP sensitivity of K1/2,ATP ∼ 1mM (data not shown), due to the open-state stabilizing effect of this mutation. As shown in Fig. 4 A, there is essentially no high affinity component of tolbutamide inhibition of Kir6.2[L164A]+SUR1 channels. On average, 100 μM tolbutamide inhibited wild-type Kir6.2+SUR1, Kir6.2[L164A]+SUR1, and Kir6.2[R176A]+SUR1 channels by 33 ± 3, 3 ± 2, and 77 ± 6%, respectively (n = 3 in each case).

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