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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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Tolbutamide sensitivity of KATP currents from cells coexpressing Kir6.2, Kir6.2[ΔN2-30], or Kir6.2[K185Q] mutant subunits and SUR1. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution (see methods). Patches were exposed to differing [tolbutamide] or 10 mM ATP, as shown. (B) Steady state dependence of membrane current on [tolbutamide] [mean ± SEM, relative to current in zero tolbutamide (Irel)] for wild-type and mutant channels (from records such as those shown in Fig. 1 A). Data points represent the mean ± SEM (n = 3–8 patches). For all channels, the lines are fits of the sum of two Hill components (as in Gribble et al. 1997a), each of the form {Irel = 1/[1 + ([tolbutamide]/K1/2)H]} with H fixed at 1.3 in each case, and the K1/2 = 2 μM (high affinity) and 4.2 mM (low affinity). The relative fraction of each component was varied. The high-affinity component was 40, 35, and 7% for wild-type, Kir6.2[K185Q]/SUR1, and Kir6.2[K185Q]/SUR1 channels, respectively.
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Figure 1: Tolbutamide sensitivity of KATP currents from cells coexpressing Kir6.2, Kir6.2[ΔN2-30], or Kir6.2[K185Q] mutant subunits and SUR1. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution (see methods). Patches were exposed to differing [tolbutamide] or 10 mM ATP, as shown. (B) Steady state dependence of membrane current on [tolbutamide] [mean ± SEM, relative to current in zero tolbutamide (Irel)] for wild-type and mutant channels (from records such as those shown in Fig. 1 A). Data points represent the mean ± SEM (n = 3–8 patches). For all channels, the lines are fits of the sum of two Hill components (as in Gribble et al. 1997a), each of the form {Irel = 1/[1 + ([tolbutamide]/K1/2)H]} with H fixed at 1.3 in each case, and the K1/2 = 2 μM (high affinity) and 4.2 mM (low affinity). The relative fraction of each component was varied. The high-affinity component was 40, 35, and 7% for wild-type, Kir6.2[K185Q]/SUR1, and Kir6.2[K185Q]/SUR1 channels, respectively.

Mentions: Gribble et al. 1997a reported that tolbutamide inhibition of Kir6.2+SUR1 coexpressed channels in Xenopus oocytes is biphasic, consisting of low and high affinity components. The mechanistic basis of the biphasic response to tolbutamide is presently unknown (see discussion), but it is clear that high affinity sulfonylurea interaction is with the SUR1 subunit (Aguilar-Bryan et al. 1995), whereas a low affinity action may occur through direct interaction with the Kir6.2 subunit (Gribble et al. 1997a). As shown in Fig. 1, similar biphasic dose–response curves are seen for both wild-type Kir6.2+SUR1 (WT+SUR1) channels and for Kir6.2[K185Q]+SUR1 channels expressed in COSm6 cells. The K185Q mutation in Kir6.2 reduces ATP sensitivity, possibly by altering ATP binding affinity, but does not affect the ATP-independent open probability (Tucker et al. 1997; Koster et al. 1999). In contrast, Kir6.2[ΔN2-30]+SUR1 channels also have a reduced ATP sensitivity, which in this case results from open-state stabilization that is reflected by near continuous bursting at the single channel level (Koster et al. 1999), and these channels show only low affinity inhibition by tolbutamide (Fig. 1 A). This raises alternate possibilities that high affinity tolbutamide block is lacking from Kir6.2[ΔN2-30] channels because the NH2 terminus is physically involved in “coupling” to the regulatory effects of SUR1, or because the high affinity inhibitory effect of tolbutamide depends on channel open state stability.


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)

Tolbutamide sensitivity of KATP currents from cells coexpressing Kir6.2, Kir6.2[ΔN2-30], or Kir6.2[K185Q] mutant subunits and SUR1. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution (see methods). Patches were exposed to differing [tolbutamide] or 10 mM ATP, as shown. (B) Steady state dependence of membrane current on [tolbutamide] [mean ± SEM, relative to current in zero tolbutamide (Irel)] for wild-type and mutant channels (from records such as those shown in Fig. 1 A). Data points represent the mean ± SEM (n = 3–8 patches). For all channels, the lines are fits of the sum of two Hill components (as in Gribble et al. 1997a), each of the form {Irel = 1/[1 + ([tolbutamide]/K1/2)H]} with H fixed at 1.3 in each case, and the K1/2 = 2 μM (high affinity) and 4.2 mM (low affinity). The relative fraction of each component was varied. The high-affinity component was 40, 35, and 7% for wild-type, Kir6.2[K185Q]/SUR1, and Kir6.2[K185Q]/SUR1 channels, respectively.
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Figure 1: Tolbutamide sensitivity of KATP currents from cells coexpressing Kir6.2, Kir6.2[ΔN2-30], or Kir6.2[K185Q] mutant subunits and SUR1. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant KATP channels at −50 mV in Kint solution (see methods). Patches were exposed to differing [tolbutamide] or 10 mM ATP, as shown. (B) Steady state dependence of membrane current on [tolbutamide] [mean ± SEM, relative to current in zero tolbutamide (Irel)] for wild-type and mutant channels (from records such as those shown in Fig. 1 A). Data points represent the mean ± SEM (n = 3–8 patches). For all channels, the lines are fits of the sum of two Hill components (as in Gribble et al. 1997a), each of the form {Irel = 1/[1 + ([tolbutamide]/K1/2)H]} with H fixed at 1.3 in each case, and the K1/2 = 2 μM (high affinity) and 4.2 mM (low affinity). The relative fraction of each component was varied. The high-affinity component was 40, 35, and 7% for wild-type, Kir6.2[K185Q]/SUR1, and Kir6.2[K185Q]/SUR1 channels, respectively.
Mentions: Gribble et al. 1997a reported that tolbutamide inhibition of Kir6.2+SUR1 coexpressed channels in Xenopus oocytes is biphasic, consisting of low and high affinity components. The mechanistic basis of the biphasic response to tolbutamide is presently unknown (see discussion), but it is clear that high affinity sulfonylurea interaction is with the SUR1 subunit (Aguilar-Bryan et al. 1995), whereas a low affinity action may occur through direct interaction with the Kir6.2 subunit (Gribble et al. 1997a). As shown in Fig. 1, similar biphasic dose–response curves are seen for both wild-type Kir6.2+SUR1 (WT+SUR1) channels and for Kir6.2[K185Q]+SUR1 channels expressed in COSm6 cells. The K185Q mutation in Kir6.2 reduces ATP sensitivity, possibly by altering ATP binding affinity, but does not affect the ATP-independent open probability (Tucker et al. 1997; Koster et al. 1999). In contrast, Kir6.2[ΔN2-30]+SUR1 channels also have a reduced ATP sensitivity, which in this case results from open-state stabilization that is reflected by near continuous bursting at the single channel level (Koster et al. 1999), and these channels show only low affinity inhibition by tolbutamide (Fig. 1 A). This raises alternate possibilities that high affinity tolbutamide block is lacking from Kir6.2[ΔN2-30] channels because the NH2 terminus is physically involved in “coupling” to the regulatory effects of SUR1, or because the high affinity inhibitory effect of tolbutamide depends on channel open state stability.

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