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Kir6.2 variant E23K increases ATP-sensitive K+ channel activity and is associated with impaired insulin release and enhanced insulin sensitivity in adults with normal glucose tolerance.

Villareal DT, Koster JC, Robertson H, Akrouh A, Miyake K, Bell GI, Patterson BW, Nichols CG, Polonsky KS - Diabetes (2009)

Bottom Line: Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity.The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT

Objective: The E23K variant in the Kir6.2 subunit of the ATP-sensitive K(+) channel (K(ATP) channel) is associated with increased risk of type 2 diabetes. The present study was undertaken to increase our understanding of the mechanisms responsible. To avoid confounding effects of hyperglycemia, insulin secretion and action were studied in subjects with the variant who had normal glucose tolerance.

Research design and methods: Nine subjects with the E23K genotype K/K and nine matched subjects with the E/E genotype underwent 5-h oral glucose tolerance tests (OGTTs), graded glucose infusion, and hyperinsulinemic-euglycemic clamp with stable-isotope-labeled tracer infusions to assess insulin secretion, action, and clearance. A total of 461 volunteers consecutively genotyped for the E23K variant also underwent OGTTs. Functional studies of the wild-type and E23K variant potassium channels were conducted.

Results: Insulin secretory responses to oral and intravenous glucose were reduced by approximately 40% in glucose-tolerant subjects homozygous for E23K. Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity. The hyperinsulinemic-euglycemic clamp revealed that hepatic insulin sensitivity is approximately 40% greater in subjects with the E23K variant, and these subjects demonstrate increased insulin sensitivity after oral glucose. The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.

Conclusions: The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion. Initially, insulin sensitivity is enhanced, thereby maintaining normal glucose tolerance. Presumably, over time, as insulin secretion falls further or insulin resistance develops, glucose levels rise resulting in type 2 diabetes.

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Related in: MedlinePlus

An increase in maximum open probability underlies the reduced ATP-sensitivity of K/K variant channels. A: Open probability in zero ATP (Po zero) calculated using the PIP2 method and noise analysis (NA) (see research design and methods) for membrane patches expressing either homomeric E/E or K/K channels. Data points represent means ± SE (n = 21–44 patches for NA method; n = 6–8 patches for PIP2 method). *P < 0.05 and **P < 0.01 by two-tailed Student's t test assuming equal variance. B: Relationship between Po zero (calculated using the NA method) and ATP sensitivity (K1/2 ATP) for individual membrane patches expressing E/E or K/K channels together with averaged data (triangles). For E/E: K1/2 ATP = 9.4 ± 1.6 μmol/l, Po zero = 0.49 ± 0.05 (n = 18 patches). For K/K: K1/2 ATP = 21.6 ± 3.3 μmol/l, Po zero = 0.0.68 ± 0.04 (n = 18 patches). **P < 0.01 for both K1/2 ATP and Po zero values of E/E compared with K/K channels (unpaired Student's t test). Data points represent means ± SE. C: Relationship between Po zero and K1/2 ATP (mmol/l). Solid line represents prediction of kinetic model II (inset) of Enkvetchakul et al. (29). Symbols represent data points as in B together with mean values for Q52R and I296L channels. The key feature of the model is that ATP acts by binding to a closed state and in consequence ATP sensitivity is reduced by shifting the equilibrium between the Cin and O states toward the open state (increasing KCO).
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Figure 5: An increase in maximum open probability underlies the reduced ATP-sensitivity of K/K variant channels. A: Open probability in zero ATP (Po zero) calculated using the PIP2 method and noise analysis (NA) (see research design and methods) for membrane patches expressing either homomeric E/E or K/K channels. Data points represent means ± SE (n = 21–44 patches for NA method; n = 6–8 patches for PIP2 method). *P < 0.05 and **P < 0.01 by two-tailed Student's t test assuming equal variance. B: Relationship between Po zero (calculated using the NA method) and ATP sensitivity (K1/2 ATP) for individual membrane patches expressing E/E or K/K channels together with averaged data (triangles). For E/E: K1/2 ATP = 9.4 ± 1.6 μmol/l, Po zero = 0.49 ± 0.05 (n = 18 patches). For K/K: K1/2 ATP = 21.6 ± 3.3 μmol/l, Po zero = 0.0.68 ± 0.04 (n = 18 patches). **P < 0.01 for both K1/2 ATP and Po zero values of E/E compared with K/K channels (unpaired Student's t test). Data points represent means ± SE. C: Relationship between Po zero and K1/2 ATP (mmol/l). Solid line represents prediction of kinetic model II (inset) of Enkvetchakul et al. (29). Symbols represent data points as in B together with mean values for Q52R and I296L channels. The key feature of the model is that ATP acts by binding to a closed state and in consequence ATP sensitivity is reduced by shifting the equilibrium between the Cin and O states toward the open state (increasing KCO).

Mentions: Kir6.2 mutations can reduce ATP sensitivity by directly reducing ATP binding to the Kir6.2 subunit or indirectly by affecting the intrinsic opening ability (28,29,34). In the latter case, an increase in the open probability (Po,zero) decreases the frequency with which the channel enters the ATP-accessible closed state, resulting in a decrease in ATP sensitivity. We have modeled this nonlinear relationship between Po,zero and K1/2,ATP (29), and this kinetic model describes the diabetes-causing effects of Kir6.2 mutations that underlie NDM (e.g., Q52R, I296L) (Fig. 5C). To estimate the open probability of KATP channels, both nonstationary noise analysis (NA) and phosphatidylinositol biphosphate (PIP2) application were used to independently examine KATP channel gating in isolated membrane patches. As shown in Fig. 5A, the estimated open probability (Po,zero) of K/K channels in the absence of ATP (0.68 ± 0.04 [NA method]; 0.67 ± 0.08 [PIP2 method]) is higher than that of E/E channels (0.49 ± 0.05 [NA method]; 0.46 ± 0.06 [PIP2 method], P < 0.05), and this increase can fully account for the shifted ATP sensitivity (K1/2,ATP = 9.4 ± 1.6 μmol/l [E/E] and 21.6 ± 3.3 μmol/l [K/K] from curve fit of individual membrane patches, P < 0.01) (Fig. 5B). A similar increase in open probability was reported for K/K channels by Schwanstecher et al. (5), using analysis of single-channel records. That multiple methodologies reiterate the same findings strengthens the conclusion that the changes associated with the E23K variant are significant and real. As with more severe NDM, the E23K variant indirectly affects ATP sensitivity by increasing the Po,zero (Fig. 5C). The predicted consequence will be reduced excitability of the β-cell, with increasingly severe consequences for insulin secretion (E23K < Q52R < I296L).


Kir6.2 variant E23K increases ATP-sensitive K+ channel activity and is associated with impaired insulin release and enhanced insulin sensitivity in adults with normal glucose tolerance.

Villareal DT, Koster JC, Robertson H, Akrouh A, Miyake K, Bell GI, Patterson BW, Nichols CG, Polonsky KS - Diabetes (2009)

An increase in maximum open probability underlies the reduced ATP-sensitivity of K/K variant channels. A: Open probability in zero ATP (Po zero) calculated using the PIP2 method and noise analysis (NA) (see research design and methods) for membrane patches expressing either homomeric E/E or K/K channels. Data points represent means ± SE (n = 21–44 patches for NA method; n = 6–8 patches for PIP2 method). *P < 0.05 and **P < 0.01 by two-tailed Student's t test assuming equal variance. B: Relationship between Po zero (calculated using the NA method) and ATP sensitivity (K1/2 ATP) for individual membrane patches expressing E/E or K/K channels together with averaged data (triangles). For E/E: K1/2 ATP = 9.4 ± 1.6 μmol/l, Po zero = 0.49 ± 0.05 (n = 18 patches). For K/K: K1/2 ATP = 21.6 ± 3.3 μmol/l, Po zero = 0.0.68 ± 0.04 (n = 18 patches). **P < 0.01 for both K1/2 ATP and Po zero values of E/E compared with K/K channels (unpaired Student's t test). Data points represent means ± SE. C: Relationship between Po zero and K1/2 ATP (mmol/l). Solid line represents prediction of kinetic model II (inset) of Enkvetchakul et al. (29). Symbols represent data points as in B together with mean values for Q52R and I296L channels. The key feature of the model is that ATP acts by binding to a closed state and in consequence ATP sensitivity is reduced by shifting the equilibrium between the Cin and O states toward the open state (increasing KCO).
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Figure 5: An increase in maximum open probability underlies the reduced ATP-sensitivity of K/K variant channels. A: Open probability in zero ATP (Po zero) calculated using the PIP2 method and noise analysis (NA) (see research design and methods) for membrane patches expressing either homomeric E/E or K/K channels. Data points represent means ± SE (n = 21–44 patches for NA method; n = 6–8 patches for PIP2 method). *P < 0.05 and **P < 0.01 by two-tailed Student's t test assuming equal variance. B: Relationship between Po zero (calculated using the NA method) and ATP sensitivity (K1/2 ATP) for individual membrane patches expressing E/E or K/K channels together with averaged data (triangles). For E/E: K1/2 ATP = 9.4 ± 1.6 μmol/l, Po zero = 0.49 ± 0.05 (n = 18 patches). For K/K: K1/2 ATP = 21.6 ± 3.3 μmol/l, Po zero = 0.0.68 ± 0.04 (n = 18 patches). **P < 0.01 for both K1/2 ATP and Po zero values of E/E compared with K/K channels (unpaired Student's t test). Data points represent means ± SE. C: Relationship between Po zero and K1/2 ATP (mmol/l). Solid line represents prediction of kinetic model II (inset) of Enkvetchakul et al. (29). Symbols represent data points as in B together with mean values for Q52R and I296L channels. The key feature of the model is that ATP acts by binding to a closed state and in consequence ATP sensitivity is reduced by shifting the equilibrium between the Cin and O states toward the open state (increasing KCO).
Mentions: Kir6.2 mutations can reduce ATP sensitivity by directly reducing ATP binding to the Kir6.2 subunit or indirectly by affecting the intrinsic opening ability (28,29,34). In the latter case, an increase in the open probability (Po,zero) decreases the frequency with which the channel enters the ATP-accessible closed state, resulting in a decrease in ATP sensitivity. We have modeled this nonlinear relationship between Po,zero and K1/2,ATP (29), and this kinetic model describes the diabetes-causing effects of Kir6.2 mutations that underlie NDM (e.g., Q52R, I296L) (Fig. 5C). To estimate the open probability of KATP channels, both nonstationary noise analysis (NA) and phosphatidylinositol biphosphate (PIP2) application were used to independently examine KATP channel gating in isolated membrane patches. As shown in Fig. 5A, the estimated open probability (Po,zero) of K/K channels in the absence of ATP (0.68 ± 0.04 [NA method]; 0.67 ± 0.08 [PIP2 method]) is higher than that of E/E channels (0.49 ± 0.05 [NA method]; 0.46 ± 0.06 [PIP2 method], P < 0.05), and this increase can fully account for the shifted ATP sensitivity (K1/2,ATP = 9.4 ± 1.6 μmol/l [E/E] and 21.6 ± 3.3 μmol/l [K/K] from curve fit of individual membrane patches, P < 0.01) (Fig. 5B). A similar increase in open probability was reported for K/K channels by Schwanstecher et al. (5), using analysis of single-channel records. That multiple methodologies reiterate the same findings strengthens the conclusion that the changes associated with the E23K variant are significant and real. As with more severe NDM, the E23K variant indirectly affects ATP sensitivity by increasing the Po,zero (Fig. 5C). The predicted consequence will be reduced excitability of the β-cell, with increasingly severe consequences for insulin secretion (E23K < Q52R < I296L).

Bottom Line: Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity.The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT

Objective: The E23K variant in the Kir6.2 subunit of the ATP-sensitive K(+) channel (K(ATP) channel) is associated with increased risk of type 2 diabetes. The present study was undertaken to increase our understanding of the mechanisms responsible. To avoid confounding effects of hyperglycemia, insulin secretion and action were studied in subjects with the variant who had normal glucose tolerance.

Research design and methods: Nine subjects with the E23K genotype K/K and nine matched subjects with the E/E genotype underwent 5-h oral glucose tolerance tests (OGTTs), graded glucose infusion, and hyperinsulinemic-euglycemic clamp with stable-isotope-labeled tracer infusions to assess insulin secretion, action, and clearance. A total of 461 volunteers consecutively genotyped for the E23K variant also underwent OGTTs. Functional studies of the wild-type and E23K variant potassium channels were conducted.

Results: Insulin secretory responses to oral and intravenous glucose were reduced by approximately 40% in glucose-tolerant subjects homozygous for E23K. Normal glucose tolerance with reduced insulin secretion suggests a change in insulin sensitivity. The hyperinsulinemic-euglycemic clamp revealed that hepatic insulin sensitivity is approximately 40% greater in subjects with the E23K variant, and these subjects demonstrate increased insulin sensitivity after oral glucose. The reconstituted E23K channels confirm reduced sensitivity to inhibitory ATP and increase in open probability, a direct molecular explanation for reduced insulin secretion.

Conclusions: The E23K variant leads to overactivity of the K(ATP) channel, resulting in reduced insulin secretion. Initially, insulin sensitivity is enhanced, thereby maintaining normal glucose tolerance. Presumably, over time, as insulin secretion falls further or insulin resistance develops, glucose levels rise resulting in type 2 diabetes.

Show MeSH
Related in: MedlinePlus