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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.

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Pharmacological characterization of voltage-gated K+-currents. A: Current responses recorded during depolarizations to +20 mV under control conditions, after addition of 10 mmol/l TEA (gray trace) and after addition of 5 mmol/l 4-aminopyridine in the continued presence of TEA (n = 4). B: As in A but pulse went to zero and currents were recorded in the absence and presence of stromatoxin (100 nmol/l, n = 4). C: As in A but pulse went to −10 mV and currents were recorded in the presence of 10 mmol/l TEA before and after application of heteropodatoxin-2 (0.5 μmol/l). D: Steady-state inactivation of the A-current analyzed by a two-pulse protocol consisting of a 200-ms conditioning pulse to membrane potentials between −90 and −20 mV followed by a 100-ms test pulse to +30 mV after an interval of 10 ms. Experiments were performed using the perforated-patch technique in the presence of TEA. E: Steady-state inactivation of the delayed-rectifying K+-current was measured by applying 15-s conditioning pulses to membrane potentials between −60 and −20 mV followed by a 500-ms test pulse to +20 mV (interval 10 ms). F: Voltage dependence of inactivation of A-current (closed circles) and delayed-rectifier current (open circles). The responses after conditioning pulses to −90 and −60 mV, respectively, were taken as unity, and data are presented as a fraction of the maximal current displayed against the voltage during the conditioning pulse. A Boltzmann function has been fit to the data points (n = 5–7). G: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of 0.5 μmol/l heteropodatoxin-2 at 1 or 6 mmol/l glucose. *P < 0.01 versus 1 mmol/l glucose alone. 100% = 6.5 ± 0.8 pg/islet/h (n = 12; 4 donors). H: Effects of 100 nmol/l stromatoxin on glucagon secretion at 1 or 20 mmol/l glucose. *P < 0.05 versus 1 mmol/l glucose. 100% = 7.5 ± 1.5 pg/islet/h (n = 9; 3 donors).
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Figure 3: Pharmacological characterization of voltage-gated K+-currents. A: Current responses recorded during depolarizations to +20 mV under control conditions, after addition of 10 mmol/l TEA (gray trace) and after addition of 5 mmol/l 4-aminopyridine in the continued presence of TEA (n = 4). B: As in A but pulse went to zero and currents were recorded in the absence and presence of stromatoxin (100 nmol/l, n = 4). C: As in A but pulse went to −10 mV and currents were recorded in the presence of 10 mmol/l TEA before and after application of heteropodatoxin-2 (0.5 μmol/l). D: Steady-state inactivation of the A-current analyzed by a two-pulse protocol consisting of a 200-ms conditioning pulse to membrane potentials between −90 and −20 mV followed by a 100-ms test pulse to +30 mV after an interval of 10 ms. Experiments were performed using the perforated-patch technique in the presence of TEA. E: Steady-state inactivation of the delayed-rectifying K+-current was measured by applying 15-s conditioning pulses to membrane potentials between −60 and −20 mV followed by a 500-ms test pulse to +20 mV (interval 10 ms). F: Voltage dependence of inactivation of A-current (closed circles) and delayed-rectifier current (open circles). The responses after conditioning pulses to −90 and −60 mV, respectively, were taken as unity, and data are presented as a fraction of the maximal current displayed against the voltage during the conditioning pulse. A Boltzmann function has been fit to the data points (n = 5–7). G: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of 0.5 μmol/l heteropodatoxin-2 at 1 or 6 mmol/l glucose. *P < 0.01 versus 1 mmol/l glucose alone. 100% = 6.5 ± 0.8 pg/islet/h (n = 12; 4 donors). H: Effects of 100 nmol/l stromatoxin on glucagon secretion at 1 or 20 mmol/l glucose. *P < 0.05 versus 1 mmol/l glucose. 100% = 7.5 ± 1.5 pg/islet/h (n = 9; 3 donors).

Mentions: The broad-spectrum K+-channel blocker tetraethylammonium (TEA) (10 mmol/l) inhibited 74 ± 2% (n = 6; P < 0.01) of the peak current and 87 ± 4% (n = 6; P < 0.01) of the sustained current evoked by depolarizations to +30 mV (Fig. 3A). The TEA-resistant transient component was completely blocked by 4-aminopyridine (5 mmol/l, n = 4; Fig. 2A). These pharmacological properties are those expected for A-type K+-currents (A-current) (25). The selective KV2.1/2.2 channel blocker stromatoxin (26) reduced the sustained current by 88 ± 5% (P < 0.01, n = 4) but decreased the peak current by only 33 ± 15% (P = 0.05; Fig. 3B). During depolarizations to 0 mV, the TEA- and stromatoxin-resistant A-current underwent rapid activation and inactivation. In seven different cells, the time constants of activation (τn) and inactivation (τh) averaged 0.26 ± 0.06 and 12 ± 3 ms, respectively (τn and τh were estimated assuming n4h kinetics). τn decreased with increasing voltages (reflecting more rapid activation), whereas no clear voltage dependence of τh was observed (not shown). The A-current was sensitive to the selective KV4.x-antagonist heteropodatoxin-2 (27) (Fig. 3C; n = 3).


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)

Pharmacological characterization of voltage-gated K+-currents. A: Current responses recorded during depolarizations to +20 mV under control conditions, after addition of 10 mmol/l TEA (gray trace) and after addition of 5 mmol/l 4-aminopyridine in the continued presence of TEA (n = 4). B: As in A but pulse went to zero and currents were recorded in the absence and presence of stromatoxin (100 nmol/l, n = 4). C: As in A but pulse went to −10 mV and currents were recorded in the presence of 10 mmol/l TEA before and after application of heteropodatoxin-2 (0.5 μmol/l). D: Steady-state inactivation of the A-current analyzed by a two-pulse protocol consisting of a 200-ms conditioning pulse to membrane potentials between −90 and −20 mV followed by a 100-ms test pulse to +30 mV after an interval of 10 ms. Experiments were performed using the perforated-patch technique in the presence of TEA. E: Steady-state inactivation of the delayed-rectifying K+-current was measured by applying 15-s conditioning pulses to membrane potentials between −60 and −20 mV followed by a 500-ms test pulse to +20 mV (interval 10 ms). F: Voltage dependence of inactivation of A-current (closed circles) and delayed-rectifier current (open circles). The responses after conditioning pulses to −90 and −60 mV, respectively, were taken as unity, and data are presented as a fraction of the maximal current displayed against the voltage during the conditioning pulse. A Boltzmann function has been fit to the data points (n = 5–7). G: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of 0.5 μmol/l heteropodatoxin-2 at 1 or 6 mmol/l glucose. *P < 0.01 versus 1 mmol/l glucose alone. 100% = 6.5 ± 0.8 pg/islet/h (n = 12; 4 donors). H: Effects of 100 nmol/l stromatoxin on glucagon secretion at 1 or 20 mmol/l glucose. *P < 0.05 versus 1 mmol/l glucose. 100% = 7.5 ± 1.5 pg/islet/h (n = 9; 3 donors).
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Related In: Results  -  Collection

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Figure 3: Pharmacological characterization of voltage-gated K+-currents. A: Current responses recorded during depolarizations to +20 mV under control conditions, after addition of 10 mmol/l TEA (gray trace) and after addition of 5 mmol/l 4-aminopyridine in the continued presence of TEA (n = 4). B: As in A but pulse went to zero and currents were recorded in the absence and presence of stromatoxin (100 nmol/l, n = 4). C: As in A but pulse went to −10 mV and currents were recorded in the presence of 10 mmol/l TEA before and after application of heteropodatoxin-2 (0.5 μmol/l). D: Steady-state inactivation of the A-current analyzed by a two-pulse protocol consisting of a 200-ms conditioning pulse to membrane potentials between −90 and −20 mV followed by a 100-ms test pulse to +30 mV after an interval of 10 ms. Experiments were performed using the perforated-patch technique in the presence of TEA. E: Steady-state inactivation of the delayed-rectifying K+-current was measured by applying 15-s conditioning pulses to membrane potentials between −60 and −20 mV followed by a 500-ms test pulse to +20 mV (interval 10 ms). F: Voltage dependence of inactivation of A-current (closed circles) and delayed-rectifier current (open circles). The responses after conditioning pulses to −90 and −60 mV, respectively, were taken as unity, and data are presented as a fraction of the maximal current displayed against the voltage during the conditioning pulse. A Boltzmann function has been fit to the data points (n = 5–7). G: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of 0.5 μmol/l heteropodatoxin-2 at 1 or 6 mmol/l glucose. *P < 0.01 versus 1 mmol/l glucose alone. 100% = 6.5 ± 0.8 pg/islet/h (n = 12; 4 donors). H: Effects of 100 nmol/l stromatoxin on glucagon secretion at 1 or 20 mmol/l glucose. *P < 0.05 versus 1 mmol/l glucose. 100% = 7.5 ± 1.5 pg/islet/h (n = 9; 3 donors).
Mentions: The broad-spectrum K+-channel blocker tetraethylammonium (TEA) (10 mmol/l) inhibited 74 ± 2% (n = 6; P < 0.01) of the peak current and 87 ± 4% (n = 6; P < 0.01) of the sustained current evoked by depolarizations to +30 mV (Fig. 3A). The TEA-resistant transient component was completely blocked by 4-aminopyridine (5 mmol/l, n = 4; Fig. 2A). These pharmacological properties are those expected for A-type K+-currents (A-current) (25). The selective KV2.1/2.2 channel blocker stromatoxin (26) reduced the sustained current by 88 ± 5% (P < 0.01, n = 4) but decreased the peak current by only 33 ± 15% (P = 0.05; Fig. 3B). During depolarizations to 0 mV, the TEA- and stromatoxin-resistant A-current underwent rapid activation and inactivation. In seven different cells, the time constants of activation (τn) and inactivation (τh) averaged 0.26 ± 0.06 and 12 ± 3 ms, respectively (τn and τh were estimated assuming n4h kinetics). τn decreased with increasing voltages (reflecting more rapid activation), whereas no clear voltage dependence of τh was observed (not shown). The A-current was sensitive to the selective KV4.x-antagonist heteropodatoxin-2 (27) (Fig. 3C; n = 3).

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