CaV2.3 calcium channels control second-phase insulin release

Abstract

Concerted activation of different voltage-gated Ca( (2+) ) channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca(V)2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca(V)2.3-knockout (Ca(V)2.3(-/-)) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca(2+) channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca(V)2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca(2+) signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca(V)2.3(-/-) beta cells. Ca(V)2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca(V)2.3(-/-) mouse. We propose a specific role for Ca(V)2.3 Ca(2+) channels in second-phase insulin release, that of mediating the Ca(2+) entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.

Figure 1

Whole-cell Ca²⁺ currents in islet cells from WT and CaV2.3^–/– mice. (A) Whole-cell Ca²⁺ currents (i) evoked by a 300-ms voltage-clamp depolarization (V) in WT CaV2.3^+/+ (black) and CaV2.3^–/– (gray) β cells. β cells were identified by exhibiting half-maximal Na⁺ channel inactivation at membrane potentials (V) lower than –100 mV (half-maximal inactivation at –102 mV; inset). (B) Average integrated current-voltage (Q-V) relationships. Data are mean values ± SEM in 10 WT (filled circles) and 10 CaV2.3^–/– (shaded circles) β cells. *P < 0.05. (C) Whole-cell Ca²⁺ currents were recorded as in A, but using CaV2.3^–/– β cells. The recordings were made under control conditions (lower gray line) in the presence of R-type Ca²⁺ channel blocker SNX482 (100 nM; black line) and after addition of L-type Ca²⁺ channel inhibitor isradipine (isr) (2 μM; upper gray line). (D) Average Q-V relationships representing mean values ± SEM in 4 CaV2.3^–/– β cells under control conditions (shaded circles), in the presence of SNX482 (filled circles), and after addition of isradipine (open circles). *P < 0.05, **P < 0.01, control versus SNX482 plus isradipine. (E) Whole-cell Ca²⁺ currents were recorded as in A, but in α cells identified by Na⁺ channel inactivation at membrane potentials greater than –100 mV (half-maximal inactivation at –49 mV; inset). (F) Q-V relationships in α cells. Data represent average values ± SEM in 8 WT (filled circles) and 4 CaV2.3^–/– (shaded circles) α cells. pC, picocoulombs.

Figure 2

Effects of CaV2.3 ablation on single-cell exocytosis in islet cells. (A) Exocytosis evoked by trains of 10 depolarizations (V) and monitored as increases in cell capacitance (ØC) in WT CaV2.3^+/+ (black) and CaV2.3^–/– (gray) β cells. (B) Average total increase in capacitance evoked by the trains (ØC_TOT). Data are mean values ± SEM in 6 WT (black bars) and 7 CaV2.3^–/– (gray bars) β cells. *P < 0.05. (C) ØC evoked by intracellular dialysis of a Ca²⁺-containing patch electrode solution (free [Ca²⁺]_i, approximately 1.5 μM) in WT (black) and CaV2.3^–/– (gray) β cells. (D) Average rates of exocytosis (ØC/Øt) ± SEM evoked by Ca²⁺ dialysis in 10 WT (black bars) and 10 CaV2.3^–/– (gray bars) β cells. (E and F) Exocytosis and average ØC were recorded as in A and B, but results are from α cells, and averages represent 5 WT (black) and 3 CaV2.3^–/– (gray) α cells. (G and H) ΔC and average rates of exocytosis were recorded as in C and D, but the data are from α cells, and mean responses are from 6 WT (black) and 3 CaV2.3^–/– (gray) α cells. pF, picofarads.

Figure 3

Ca²⁺ homeostasis in WT and CaV2.3^–/– islets. (A) [Ca²⁺]i in an intact WT CaV2.3^+/+ islet assayed by ratiometric fura-2 measurements. The islets were stimulated at the time points indicated by the arrows in the continued presence of previously added stimuli. (B) [Ca²⁺]i was determined as in A, but the experiment was performed in an intact CaV2.3^–/– islet. Recordings selected for display are representative of 7 and 9 separate experiments in WT and CaV2.3^–/– islets, respectively. Statistical significances are provided in the text.

Figure 4

Dynamics of insulin release. (A) Insulin release measured in WT CaV2.3^+/+ pancreata before and after increasing the glucose concentration in the perfusate from 3.3 mM to 16.7 mM at t = 11 minutes. Samples were taken at 60-second intervals, except during the first 10 minutes after increasing the glucose concentration (t = 11 to 21 minutes, as indicated by the gray bar) when the sample interval was 30 seconds. (B) Insulin release was measured as in A, but the experiments were performed in CaV2.3^–/– mice. To facilitate comparison with WT, mean values measured under that condition are indicated by the dotted line. Data in A and B represent means ± SEM from 4 and 5 experiments in WT and CaV2.3^–/– mice, respectively. (C) Insulin release was measured as in A, but the experiments were performed in NMRI mice. (D) Insulin release was measured as in C, but SNX482 (100 nM) was included in the high-glucose solution. To facilitate comparison with mean values measured in the absence of SNX482, these values are denoted by the dotted line. Data in C and D represent means ± SEM from 6 experiments performed with and without SNX482, respectively. Statistical significance is provided in Results.

Figure 5

Confocal immunocytochemistry of insulin and glucagon immunoreactivity in single islet cells. (A) Insulin and glucagon immunoreactivity in dispersed WT islet cells visualized by confocal microscopy. (B) Insulin and glucagon immunoreactivity was visualized as in A, but cells are from CaV2.3^–/– islets. (C) Relative distribution of insulin, glucagon, and double immunoreactivity (Ins⁺, Glu⁺, and Ins&glu⁺, respectively) in WT CaV2.3^+/+ (black bars) and CaV2.3^–/– (gray bars) islet cells. Data represent more than 200 cells in each group and are from 3 different experiments.