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Enhanced glucose tolerance by SK4 channel inhibition in pancreatic beta-cells.

Düfer M, Gier B, Wolpers D, Krippeit-Drews P, Ruth P, Drews G - Diabetes (2009)

Bottom Line: SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo.Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy, the Department of Pharmacology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Objective: Ca(2+)-regulated K(+) channels are involved in numerous Ca(2+)-dependent signaling pathways. In this study, we investigated whether the Ca(2+)-activated K(+) channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic beta-cell function.

Research design and methods: Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca(2+) concentration ([Ca(2+)](c)) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis.

Results: SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO beta-cells stimulated with 15 mmol/l glucose had an increased Ca(2+) action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca(2+)](c). In addition, glucose responsiveness of membrane potential, [Ca(2+)](c), and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K(+) currents and concomitant membrane hyperpolarization could be evoked in WT beta-cells by the SK4 channel opener DCEBIO (100 micromol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 micromol/l) partly inhibited K(Ca) currents and induced electrical activity at a threshold glucose concentration. In stimulated WT beta-cells, TRAM-34 further increased [Ca(2+)](c) and broadened action potentials similar to those seen in SK4-KO beta-cells. SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).

Conclusions: SK4 channels are involved in beta-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

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Related in: MedlinePlus

Genetic ablation or pharmacologic inhibition of SK4 channels influences electrical activity and [Ca2+]c of pancreatic β-cells. A: In the presence of 15 mmol/l glucose, action potential frequency was increased in SK4-KO β-cells compared with WT controls. Data are given as means ± SEM of 18 WT and 53 SK4-KO β-cells tested. B and D: Analysis of single Ca2+ action potentials in SK4-KO and WT β-cells. SK4 deficiency or blockage with the SK4 channel inhibitor TRAM-34 (1 μmol/l) resulted in action potential broadening and depolarized the plateau potential from which action potentials started. In the series with TRAM-34, the shape of action potentials before drug application was compared with action potentials 3–4 min after addition of TRAM-34. The traces were compiled by averaging action potentials of 11 experiments with SK4-KO and 12 experiments with WT β-cells. The series with TRAM-34 results from five independent experiments. C: SK4-KO β-cells stimulated with 15 mmol/l glucose display an augmented Ca2+ response compared with WT β-cells. The figure shows an overlay of two representative traces of the first increase in [Ca2+]c induced by switching glucose from 0.5 to 15 mmol/l (arrow). A total of 31 SK4-KO and 26 WT β-cells were analyzed. The values for AUCCa ± SEM of this series of experiments are summarized in the diagram. E: Blocking SK4 channels elevates [Ca2+]c in WT β-cells. β-Cells exposed to 11.1 mmol/l glucose show regular oscillations of [Ca2+]c. Addition of TRAM-34 (1 μmol/l) increased [Ca2+]c and altered the pattern of oscillations. The experiment is representative of five with similar results. The diagram summarizes the increase in the AUCCa analyzed for a time period of 4 min in the presence of TRAM-34 compared with control conditions. *P ≤ 0.05, **P < 0.001, ***P ≤ 0.001.
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Figure 1: Genetic ablation or pharmacologic inhibition of SK4 channels influences electrical activity and [Ca2+]c of pancreatic β-cells. A: In the presence of 15 mmol/l glucose, action potential frequency was increased in SK4-KO β-cells compared with WT controls. Data are given as means ± SEM of 18 WT and 53 SK4-KO β-cells tested. B and D: Analysis of single Ca2+ action potentials in SK4-KO and WT β-cells. SK4 deficiency or blockage with the SK4 channel inhibitor TRAM-34 (1 μmol/l) resulted in action potential broadening and depolarized the plateau potential from which action potentials started. In the series with TRAM-34, the shape of action potentials before drug application was compared with action potentials 3–4 min after addition of TRAM-34. The traces were compiled by averaging action potentials of 11 experiments with SK4-KO and 12 experiments with WT β-cells. The series with TRAM-34 results from five independent experiments. C: SK4-KO β-cells stimulated with 15 mmol/l glucose display an augmented Ca2+ response compared with WT β-cells. The figure shows an overlay of two representative traces of the first increase in [Ca2+]c induced by switching glucose from 0.5 to 15 mmol/l (arrow). A total of 31 SK4-KO and 26 WT β-cells were analyzed. The values for AUCCa ± SEM of this series of experiments are summarized in the diagram. E: Blocking SK4 channels elevates [Ca2+]c in WT β-cells. β-Cells exposed to 11.1 mmol/l glucose show regular oscillations of [Ca2+]c. Addition of TRAM-34 (1 μmol/l) increased [Ca2+]c and altered the pattern of oscillations. The experiment is representative of five with similar results. The diagram summarizes the increase in the AUCCa analyzed for a time period of 4 min in the presence of TRAM-34 compared with control conditions. *P ≤ 0.05, **P < 0.001, ***P ≤ 0.001.

Mentions: [Ca2+]c and electrophysiological experiments are illustrated by representative recordings. At least three different cell preparations were used for each series. Means ± SE are given in the text for the indicated number of experiments. Western blots were performed in duplicate. Statistical significance of differences was assessed by a one-sample or Student's t test for paired values; multiple comparisons were made by ANOVA followed by a Student-Newman-Keuls test. For AP characteristics, five APs of each experiment were averaged. Peak values were set to t = 0 ms, and data were analyzed every 50 ms within the preceding and following 200 ms (Fig. 1B and D). Dose-response curves of [Ca2+]c were fitted with the Hill equation. Curves were defined by the following parameters: P(D) = 1/[1 + (D50/D)c], where P(D) is the probability of glucose-induced stimulation, D50 is the dose level with 50% response probability, D is the glucose concentration (in mmol/l), and c reflects the slope of the concentration-response curve. The equation was adjusted by a maximum-likelihood procedure. A P value of <0.05 was considered significant.


Enhanced glucose tolerance by SK4 channel inhibition in pancreatic beta-cells.

Düfer M, Gier B, Wolpers D, Krippeit-Drews P, Ruth P, Drews G - Diabetes (2009)

Genetic ablation or pharmacologic inhibition of SK4 channels influences electrical activity and [Ca2+]c of pancreatic β-cells. A: In the presence of 15 mmol/l glucose, action potential frequency was increased in SK4-KO β-cells compared with WT controls. Data are given as means ± SEM of 18 WT and 53 SK4-KO β-cells tested. B and D: Analysis of single Ca2+ action potentials in SK4-KO and WT β-cells. SK4 deficiency or blockage with the SK4 channel inhibitor TRAM-34 (1 μmol/l) resulted in action potential broadening and depolarized the plateau potential from which action potentials started. In the series with TRAM-34, the shape of action potentials before drug application was compared with action potentials 3–4 min after addition of TRAM-34. The traces were compiled by averaging action potentials of 11 experiments with SK4-KO and 12 experiments with WT β-cells. The series with TRAM-34 results from five independent experiments. C: SK4-KO β-cells stimulated with 15 mmol/l glucose display an augmented Ca2+ response compared with WT β-cells. The figure shows an overlay of two representative traces of the first increase in [Ca2+]c induced by switching glucose from 0.5 to 15 mmol/l (arrow). A total of 31 SK4-KO and 26 WT β-cells were analyzed. The values for AUCCa ± SEM of this series of experiments are summarized in the diagram. E: Blocking SK4 channels elevates [Ca2+]c in WT β-cells. β-Cells exposed to 11.1 mmol/l glucose show regular oscillations of [Ca2+]c. Addition of TRAM-34 (1 μmol/l) increased [Ca2+]c and altered the pattern of oscillations. The experiment is representative of five with similar results. The diagram summarizes the increase in the AUCCa analyzed for a time period of 4 min in the presence of TRAM-34 compared with control conditions. *P ≤ 0.05, **P < 0.001, ***P ≤ 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712794&req=5

Figure 1: Genetic ablation or pharmacologic inhibition of SK4 channels influences electrical activity and [Ca2+]c of pancreatic β-cells. A: In the presence of 15 mmol/l glucose, action potential frequency was increased in SK4-KO β-cells compared with WT controls. Data are given as means ± SEM of 18 WT and 53 SK4-KO β-cells tested. B and D: Analysis of single Ca2+ action potentials in SK4-KO and WT β-cells. SK4 deficiency or blockage with the SK4 channel inhibitor TRAM-34 (1 μmol/l) resulted in action potential broadening and depolarized the plateau potential from which action potentials started. In the series with TRAM-34, the shape of action potentials before drug application was compared with action potentials 3–4 min after addition of TRAM-34. The traces were compiled by averaging action potentials of 11 experiments with SK4-KO and 12 experiments with WT β-cells. The series with TRAM-34 results from five independent experiments. C: SK4-KO β-cells stimulated with 15 mmol/l glucose display an augmented Ca2+ response compared with WT β-cells. The figure shows an overlay of two representative traces of the first increase in [Ca2+]c induced by switching glucose from 0.5 to 15 mmol/l (arrow). A total of 31 SK4-KO and 26 WT β-cells were analyzed. The values for AUCCa ± SEM of this series of experiments are summarized in the diagram. E: Blocking SK4 channels elevates [Ca2+]c in WT β-cells. β-Cells exposed to 11.1 mmol/l glucose show regular oscillations of [Ca2+]c. Addition of TRAM-34 (1 μmol/l) increased [Ca2+]c and altered the pattern of oscillations. The experiment is representative of five with similar results. The diagram summarizes the increase in the AUCCa analyzed for a time period of 4 min in the presence of TRAM-34 compared with control conditions. *P ≤ 0.05, **P < 0.001, ***P ≤ 0.001.
Mentions: [Ca2+]c and electrophysiological experiments are illustrated by representative recordings. At least three different cell preparations were used for each series. Means ± SE are given in the text for the indicated number of experiments. Western blots were performed in duplicate. Statistical significance of differences was assessed by a one-sample or Student's t test for paired values; multiple comparisons were made by ANOVA followed by a Student-Newman-Keuls test. For AP characteristics, five APs of each experiment were averaged. Peak values were set to t = 0 ms, and data were analyzed every 50 ms within the preceding and following 200 ms (Fig. 1B and D). Dose-response curves of [Ca2+]c were fitted with the Hill equation. Curves were defined by the following parameters: P(D) = 1/[1 + (D50/D)c], where P(D) is the probability of glucose-induced stimulation, D50 is the dose level with 50% response probability, D is the glucose concentration (in mmol/l), and c reflects the slope of the concentration-response curve. The equation was adjusted by a maximum-likelihood procedure. A P value of <0.05 was considered significant.

Bottom Line: SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo.Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy, the Department of Pharmacology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Objective: Ca(2+)-regulated K(+) channels are involved in numerous Ca(2+)-dependent signaling pathways. In this study, we investigated whether the Ca(2+)-activated K(+) channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic beta-cell function.

Research design and methods: Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca(2+) concentration ([Ca(2+)](c)) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis.

Results: SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO beta-cells stimulated with 15 mmol/l glucose had an increased Ca(2+) action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca(2+)](c). In addition, glucose responsiveness of membrane potential, [Ca(2+)](c), and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K(+) currents and concomitant membrane hyperpolarization could be evoked in WT beta-cells by the SK4 channel opener DCEBIO (100 micromol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 micromol/l) partly inhibited K(Ca) currents and induced electrical activity at a threshold glucose concentration. In stimulated WT beta-cells, TRAM-34 further increased [Ca(2+)](c) and broadened action potentials similar to those seen in SK4-KO beta-cells. SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).

Conclusions: SK4 channels are involved in beta-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

Show MeSH
Related in: MedlinePlus