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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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Summary of the standard pulse-length protocol data from the control (n = 17 cells; solid) and EGTA (n = 15 cells; dashed) groups.Data are means ± SEM for data pooled according to pulse-length. A: Evoked capacitance increases ΔCm for each depolarization of varying length (t). B: Evoked Ca2+ influx Q for each pulse length (t). C: ΔCm vs. Q for each pulse length. The gray lines indicate the Ca2+ current sensitivity in the control (solid) and EGTA (dashed) groups, as estimated by the linear mixed-effects model (see Fig. 2).
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pone-0103874-g001: Summary of the standard pulse-length protocol data from the control (n = 17 cells; solid) and EGTA (n = 15 cells; dashed) groups.Data are means ± SEM for data pooled according to pulse-length. A: Evoked capacitance increases ΔCm for each depolarization of varying length (t). B: Evoked Ca2+ influx Q for each pulse length (t). C: ΔCm vs. Q for each pulse length. The gray lines indicate the Ca2+ current sensitivity in the control (solid) and EGTA (dashed) groups, as estimated by the linear mixed-effects model (see Fig. 2).

Mentions: First, we were interested in investigating kinetics of exocytosis using an experimental setting used previously [8], [33]. Accordingly, single INS-1 832/13 cells were subject to capacitances measurements using the whole-cell configuration of the patch-clamp technique. Capacitance increases were evoked by the pulse-length protocol, which depolarizes the membrane potential from −70 mV to 0 mV during voltage-clamp periods of varying length. Pulses of different duration were applied in varying order, and no dependence on the order was found. The capacitance increase (ΔCm) reflecting exocytosis showed a biphasic relation to the pulse length such that the average rate of exocytosis was higher during short than during longer pulses (Fig. 1A) [12], [34]. This biphasic pattern has been suggested to be caused by depletion of IRP located near Ca2+ channels [8], [33]. However, because of Ca2+ current inactivation, the amount of Ca2+ (Q) that enters the cell during each depolarization does not have a simple relationship to pulse length (Fig. 1B). It might be that the biphasic pattern of the increase in membrane capacitance is caused by current inactivation rather than IRP depletion, and to investigate this question one should relate ΔCm to Q rather than to pulse length [18], [22]. Indeed, experiments performed on INS-1 cells has previously demonstrated that a depolarization of the same size and duration (300 ms) can give rise to large differences in Ca2+ influx measured as charge (Q). Plotting ΔCm to Q in this case gave a linear relationship [35]. Our data showed a near-linear ΔCm to Q relation (Fig. 1C), as previously observed in mouse beta- [8], [10], [20] and alpha-cells [30], [36]. In contrast, human beta-cells in situ show a nonlinear, concave ΔCm to Q relation [21].


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)

Summary of the standard pulse-length protocol data from the control (n = 17 cells; solid) and EGTA (n = 15 cells; dashed) groups.Data are means ± SEM for data pooled according to pulse-length. A: Evoked capacitance increases ΔCm for each depolarization of varying length (t). B: Evoked Ca2+ influx Q for each pulse length (t). C: ΔCm vs. Q for each pulse length. The gray lines indicate the Ca2+ current sensitivity in the control (solid) and EGTA (dashed) groups, as estimated by the linear mixed-effects model (see Fig. 2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103874-g001: Summary of the standard pulse-length protocol data from the control (n = 17 cells; solid) and EGTA (n = 15 cells; dashed) groups.Data are means ± SEM for data pooled according to pulse-length. A: Evoked capacitance increases ΔCm for each depolarization of varying length (t). B: Evoked Ca2+ influx Q for each pulse length (t). C: ΔCm vs. Q for each pulse length. The gray lines indicate the Ca2+ current sensitivity in the control (solid) and EGTA (dashed) groups, as estimated by the linear mixed-effects model (see Fig. 2).
Mentions: First, we were interested in investigating kinetics of exocytosis using an experimental setting used previously [8], [33]. Accordingly, single INS-1 832/13 cells were subject to capacitances measurements using the whole-cell configuration of the patch-clamp technique. Capacitance increases were evoked by the pulse-length protocol, which depolarizes the membrane potential from −70 mV to 0 mV during voltage-clamp periods of varying length. Pulses of different duration were applied in varying order, and no dependence on the order was found. The capacitance increase (ΔCm) reflecting exocytosis showed a biphasic relation to the pulse length such that the average rate of exocytosis was higher during short than during longer pulses (Fig. 1A) [12], [34]. This biphasic pattern has been suggested to be caused by depletion of IRP located near Ca2+ channels [8], [33]. However, because of Ca2+ current inactivation, the amount of Ca2+ (Q) that enters the cell during each depolarization does not have a simple relationship to pulse length (Fig. 1B). It might be that the biphasic pattern of the increase in membrane capacitance is caused by current inactivation rather than IRP depletion, and to investigate this question one should relate ΔCm to Q rather than to pulse length [18], [22]. Indeed, experiments performed on INS-1 cells has previously demonstrated that a depolarization of the same size and duration (300 ms) can give rise to large differences in Ca2+ influx measured as charge (Q). Plotting ΔCm to Q in this case gave a linear relationship [35]. Our data showed a near-linear ΔCm to Q relation (Fig. 1C), as previously observed in mouse beta- [8], [10], [20] and alpha-cells [30], [36]. In contrast, human beta-cells in situ show a nonlinear, concave ΔCm to Q relation [21].

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