Limits...
Membrane potential-dependent inactivation of voltage-gated ion channels in alpha-cells inhibits glucagon secretion from human islets.

Ramracheya R, Ward C, Shigeto M, Walker JN, Amisten S, Zhang Q, Johnson PR, Rorsman P, Braun M - Diabetes (2010)

Bottom Line: Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude.Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV.We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK. matthias.braun@drl.ox.ac.uk

ABSTRACT

Objective: To document the properties of the voltage-gated ion channels in human pancreatic alpha-cells and their role in glucagon release.

Research design and methods: Glucagon release was measured from intact islets. [Ca(2+)](i) was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated alpha-cells identified by immunocytochemistry.

Result: Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl(2). Glucose remained inhibitory in the presence of ZnCl(2) and after blockade of type-2 somatostatin receptors. Human alpha-cells are electrically active at 1 mmol/l glucose. Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude. Human alpha-cells contain heteropodatoxin-sensitive A-type K(+)-channels, stromatoxin-sensitive delayed rectifying K(+)-channels, tetrodotoxin-sensitive Na(+)-currents, and low-threshold T-type, isradipine-sensitive L-type, and omega-agatoxin-sensitive P/Q-type Ca(2+)-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, omega-agatoxin, and isradipine. The [Ca(2+)](i) oscillations depend principally on Ca(2+)-influx via L-type Ca(2+)-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV. Blocking P/Q-type Ca(2+)-currents abolished depolarization-evoked exocytosis.

Conclusions: Human alpha-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

Show MeSH

Related in: MedlinePlus

Analysis of voltage-gated K+-currents. A: Family of voltage-activated K+-currents (lower) evoked by depolarizing pulses from −70 mV to membrane potentials between −40 and +80 mV. Inset shows inactivation of current during a 15-s depolarization from −70 to +20 mV. B: As in A but showing the initial part of the current responses during pulses to −40, −30, −20, and −10 mV on an expanded time base (sections highlighted in gray in A). C: I–V relationship for voltage-gated K+-current (n = 8). D: Example of a cell showing a clear shoulder on the I–V at voltages between +30 and +50 mV. Data are shown for the peak current (squares), the sustained current measured at the end of the 500-ms pulse (circles), and the difference (triangles).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2927942&req=5

Figure 2: Analysis of voltage-gated K+-currents. A: Family of voltage-activated K+-currents (lower) evoked by depolarizing pulses from −70 mV to membrane potentials between −40 and +80 mV. Inset shows inactivation of current during a 15-s depolarization from −70 to +20 mV. B: As in A but showing the initial part of the current responses during pulses to −40, −30, −20, and −10 mV on an expanded time base (sections highlighted in gray in A). C: I–V relationship for voltage-gated K+-current (n = 8). D: Example of a cell showing a clear shoulder on the I–V at voltages between +30 and +50 mV. Data are shown for the peak current (squares), the sustained current measured at the end of the 500-ms pulse (circles), and the difference (triangles).

Mentions: Voltage-gated membrane currents were recorded using the standard whole-cell configuration. Outward voltage-activated K+-currents became detectable during membrane depolarizations from −70 to −30 mV and above (Fig. 2A and B). At −30 mV, the K+-current inactivated completely within ∼20 ms, whereas a sustained component was observed during depolarizations to −20 mV and above (Fig. 2B). The peak amplitude recorded during depolarizations to 0 mV averaged 349 ± 61 pA (n = 8). The inactivation of the current could be described as the sum of two exponentials with time constants (at +30 mV) of 18 ± 2 and 567 ± 147 ms (n = 7). The sustained current inactivated by >90% over 15 s (Fig. 2A, inset).


Membrane potential-dependent inactivation of voltage-gated ion channels in alpha-cells inhibits glucagon secretion from human islets.

Ramracheya R, Ward C, Shigeto M, Walker JN, Amisten S, Zhang Q, Johnson PR, Rorsman P, Braun M - Diabetes (2010)

Analysis of voltage-gated K+-currents. A: Family of voltage-activated K+-currents (lower) evoked by depolarizing pulses from −70 mV to membrane potentials between −40 and +80 mV. Inset shows inactivation of current during a 15-s depolarization from −70 to +20 mV. B: As in A but showing the initial part of the current responses during pulses to −40, −30, −20, and −10 mV on an expanded time base (sections highlighted in gray in A). C: I–V relationship for voltage-gated K+-current (n = 8). D: Example of a cell showing a clear shoulder on the I–V at voltages between +30 and +50 mV. Data are shown for the peak current (squares), the sustained current measured at the end of the 500-ms pulse (circles), and the difference (triangles).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927942&req=5

Figure 2: Analysis of voltage-gated K+-currents. A: Family of voltage-activated K+-currents (lower) evoked by depolarizing pulses from −70 mV to membrane potentials between −40 and +80 mV. Inset shows inactivation of current during a 15-s depolarization from −70 to +20 mV. B: As in A but showing the initial part of the current responses during pulses to −40, −30, −20, and −10 mV on an expanded time base (sections highlighted in gray in A). C: I–V relationship for voltage-gated K+-current (n = 8). D: Example of a cell showing a clear shoulder on the I–V at voltages between +30 and +50 mV. Data are shown for the peak current (squares), the sustained current measured at the end of the 500-ms pulse (circles), and the difference (triangles).
Mentions: Voltage-gated membrane currents were recorded using the standard whole-cell configuration. Outward voltage-activated K+-currents became detectable during membrane depolarizations from −70 to −30 mV and above (Fig. 2A and B). At −30 mV, the K+-current inactivated completely within ∼20 ms, whereas a sustained component was observed during depolarizations to −20 mV and above (Fig. 2B). The peak amplitude recorded during depolarizations to 0 mV averaged 349 ± 61 pA (n = 8). The inactivation of the current could be described as the sum of two exponentials with time constants (at +30 mV) of 18 ± 2 and 567 ± 147 ms (n = 7). The sustained current inactivated by >90% over 15 s (Fig. 2A, inset).

Bottom Line: Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude.Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV.We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK. matthias.braun@drl.ox.ac.uk

ABSTRACT

Objective: To document the properties of the voltage-gated ion channels in human pancreatic alpha-cells and their role in glucagon release.

Research design and methods: Glucagon release was measured from intact islets. [Ca(2+)](i) was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated alpha-cells identified by immunocytochemistry.

Result: Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl(2). Glucose remained inhibitory in the presence of ZnCl(2) and after blockade of type-2 somatostatin receptors. Human alpha-cells are electrically active at 1 mmol/l glucose. Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude. Human alpha-cells contain heteropodatoxin-sensitive A-type K(+)-channels, stromatoxin-sensitive delayed rectifying K(+)-channels, tetrodotoxin-sensitive Na(+)-currents, and low-threshold T-type, isradipine-sensitive L-type, and omega-agatoxin-sensitive P/Q-type Ca(2+)-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, omega-agatoxin, and isradipine. The [Ca(2+)](i) oscillations depend principally on Ca(2+)-influx via L-type Ca(2+)-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV. Blocking P/Q-type Ca(2+)-currents abolished depolarization-evoked exocytosis.

Conclusions: Human alpha-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

Show MeSH
Related in: MedlinePlus