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Calcium current inactivation rather than pool depletion explains reduced exocytotic rate with prolonged stimulation in insulin-secreting INS-1 832/13 cells.

Pedersen MG, Salunkhe VA, Svedin E, Edlund A, Eliasson L - PLoS ONE (2014)

Bottom Line: We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis.The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected.These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

View Article: PubMed Central - PubMed

Affiliation: Islet Cell Exocytosis, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden.

ABSTRACT
Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca2+-dependent insulin exocytosis with respect to pool depletion and Ca2+-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔCm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

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Related in: MedlinePlus

Demonstration of the double-pulse protocol.Single cells were depolarized from a holding potential of −70 mV to 0 mV during 50 ms. This was followed by a resting period of 100 ms and a second 50-ms depolarization from −70 mv to 0 mV (top trace). The evoked Ca2+ currents were measured and the charges for the first (Q1) and second (Q2) pulses were estimated (middle trace). In addition, the increases in membrane capacitance evoked by the first (ΔCm1) and second (ΔCm2) depolarizations were measured.
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pone-0103874-g003: Demonstration of the double-pulse protocol.Single cells were depolarized from a holding potential of −70 mV to 0 mV during 50 ms. This was followed by a resting period of 100 ms and a second 50-ms depolarization from −70 mv to 0 mV (top trace). The evoked Ca2+ currents were measured and the charges for the first (Q1) and second (Q2) pulses were estimated (middle trace). In addition, the increases in membrane capacitance evoked by the first (ΔCm1) and second (ΔCm2) depolarizations were measured.

Mentions: An alternative to the pulse-length protocol for the study of pool depletion is the double-pulse protocol [15], [39], where two depolarizations are applied separated by a short resting period, and the capacitance responses are measured. To investigate pool depletion from another angle we used this protocol and accordingly applied two 50 ms depolarizations to 0 mV from −70 mV separated by a 100 ms interval, in absence (control) and presence of 10 mM EGTA in the patch pipette (Fig. 3). Gillis et al. [39] suggested adjusting the voltage between pulses, such that calcium channel inactivation is balanced by stronger channel activation during the second pulse. However, the degree of inactivation is unknown until the experiment is performed, which complicates the choice of the voltage to apply during the second pulse.


Calcium current inactivation rather than pool depletion explains reduced exocytotic rate with prolonged stimulation in insulin-secreting INS-1 832/13 cells.

Pedersen MG, Salunkhe VA, Svedin E, Edlund A, Eliasson L - PLoS ONE (2014)

Demonstration of the double-pulse protocol.Single cells were depolarized from a holding potential of −70 mV to 0 mV during 50 ms. This was followed by a resting period of 100 ms and a second 50-ms depolarization from −70 mv to 0 mV (top trace). The evoked Ca2+ currents were measured and the charges for the first (Q1) and second (Q2) pulses were estimated (middle trace). In addition, the increases in membrane capacitance evoked by the first (ΔCm1) and second (ΔCm2) depolarizations were measured.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126658&req=5

pone-0103874-g003: Demonstration of the double-pulse protocol.Single cells were depolarized from a holding potential of −70 mV to 0 mV during 50 ms. This was followed by a resting period of 100 ms and a second 50-ms depolarization from −70 mv to 0 mV (top trace). The evoked Ca2+ currents were measured and the charges for the first (Q1) and second (Q2) pulses were estimated (middle trace). In addition, the increases in membrane capacitance evoked by the first (ΔCm1) and second (ΔCm2) depolarizations were measured.
Mentions: An alternative to the pulse-length protocol for the study of pool depletion is the double-pulse protocol [15], [39], where two depolarizations are applied separated by a short resting period, and the capacitance responses are measured. To investigate pool depletion from another angle we used this protocol and accordingly applied two 50 ms depolarizations to 0 mV from −70 mV separated by a 100 ms interval, in absence (control) and presence of 10 mM EGTA in the patch pipette (Fig. 3). Gillis et al. [39] suggested adjusting the voltage between pulses, such that calcium channel inactivation is balanced by stronger channel activation during the second pulse. However, the degree of inactivation is unknown until the experiment is performed, which complicates the choice of the voltage to apply during the second pulse.

Bottom Line: We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis.The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected.These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

View Article: PubMed Central - PubMed

Affiliation: Islet Cell Exocytosis, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden.

ABSTRACT
Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca2+-dependent insulin exocytosis with respect to pool depletion and Ca2+-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔCm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

Show MeSH
Related in: MedlinePlus