Potentiation of exocytosis by phospholipase C-coupled G-protein-coupled receptors requires the priming protein Munc13-1

Abstract

The vesicle priming protein Munc13-1 is regulated by diacylglycerol (DAG) and is therefore hypothesized to play a role in the control of neurotransmitter release by phospholipase C (PLC)-coupled receptors. We combined voltage-clamp recordings of voltage-gated Ca2+ channels (VGCCs) and high-resolution capacitance measurements to investigate the mechanism of receptor-mediated modulation of exocytosis in bovine chromaffin cells. Activation of endogenous H1 G(q)-protein-coupled receptors (G(q)PCRs) by histamine potentiated stimulus-coupled secretion despite concurrently inhibiting Ca2+ influx through VGCCs. Histamine increased the size of the readily releasable pool of vesicles and in particular a subpool of fusion-competent vesicles localized in close proximity to VGCCs. Pharmacological characterization showed that potentiation of exocytosis depended on the activation of PLC but not protein kinase C. Overexpression of wild-type Munc13-1 by adenoviral infection had no effect on histamine-induced potentiation per se, whereas DAG-insensitive Munc13-1(H567K) completely abolished it. This is the first endogenous mammalian G(q)PCR signaling pathway identified that engages Munc13-1 to increase stimulus-coupled secretion by recruiting vesicles to the immediately releasable pool. G(q)PCRs are therefore able to control exocytosis at the level of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation to produce rapid, short-term potentiation of the secretory output of neurons and endocrine cells.

Figure 1.

Histamine potentiates stimulus-coupled exocytosis in bovine chromaffin cells. A, Representative traces of I _Ca (top) evoked by depolarizations before (ctrl) and during superfusion of histamine (100 μm). Corresponding membrane capacitance (ΔC _m) changes (bottom) measured in the same cell in response to the Ca²⁺ entry evoked by the depolarizations. Capacitance detection was interrupted during depolarization (gap in ΔC _m trace), and ΔC _m was quantified relative to a 100 fF calibration step (indicated by ♦). B, C, Mean data ± SEM from n = 13 cells summarizing the effect of histamine (100 μm) on Ca²⁺ entry (B) and exocytotic efficiency (C). Data were normalized to control measurements made before application of histamine. The effects of histamine were completely reversed during washout of the agonist. **p < 0.01.

Figure 2.

Histamine increases the size of the RRP of vesicles. A, Sample traces of I _Ca (top) evoked by the double pulse protocol illustrated used to measure the RRP (100 ms interpulse interval). Corresponding ΔC _m1 and ΔC _m2 (bottom) measured in the same cell before and during application of histamine (100 μm). B, Sample traces of ΔC _m evoked by a train of 6 × 10 ms (390 ms interpulse interval) followed by 4 × 100 ms depolarizations (300 ms interpulse interval) measured in the same cell before and during application of histamine (100 μm). C, Mean ± SEM from n = 10 cells summarizing the effect of histamine on the exocytotic efficiency of a train of 6 × 10 ms followed by 4 × 100 ms depolarizations. The exocytotic efficiency was calculated as ΣΔC _m/Σ∫Ca²⁺ for stimulus 1–6 and stimulus 7–10. **p < 0.01.

Figure 3.

PKC is not involved in histamine potentiation. A, Mean data of normalized exocytotic efficiency measured in the presence of histamine (100 μm; n = 19), calphostin C (Cal) (100 nm) plus histamine (100 μm; n = 6), and Bis (0.5–1 μm) plus histamine (n = 7). B, Inset, Overlaid sample traces of I _Ca before (ctrl) and 4 min into PMA (250 nm) application. Bar graph, Mean ± SEM percentage change in peak I _Ca by PMA (250 nm) in untreated (n = 6) and Bis (0.5–1 μm)-treated cells (n = 7). Note that the experiment represented in B was performed on the same cells represented in A and confirms the effectiveness of the Bis pretreatment. **p < 0.01.

Figure 4.

Exogenous gene expression in bovine chromaffin cells using adenovirus. A, Mean ± SEM peak I _Ca (top) and corresponding membrane capacitance changes (ΔC _m) (bottom) evoked by a 200 ms depolarizing pulse from a holding potential of −80 to +20 mV in noninfected cells (n = 15) and culture-matched cells infected with adenovirus for EGFP expression (n = 11). B, Mean ± SEM stimulus-coupled synchronous (s) and poststimulus asynchronous (as) ΔC _m in noninfected (black filled bar; n = 9) and culture-matched EGFP-expressing cells (gray filled bar; n = 12) evoked by a train of 10 depolarizations (550 ms interpulse interval). C, Mean ± SEM of ΔC _m evoked by stimulus 1–6 (secretion from the IRP) and stimulus 7–10 (remaining RRP and part of SRP) in noninfected (black filled bar; n = 5) and culture-matched EGFP-expressing cells (gray filled bar; n = 5). Cells were stimulated by a train of 6 × 10 ms (390 ms interpulse interval) and 4 × 100 ms depolarizing pulses (300 ms interpulse interval). D, Sample traces of fura-2 measurements (R _340/380; top) and corresponding ΔC _m (bottom) evoked by 50 ms depolarizing pulses from −80 to +20 mV (indicated by downward arrows) recorded in a cell expressing CaM₁₂₃₄/EGFP. The dashed line indicates basal Ca²⁺ levels. A 100 fF calibration step (indicated by ♦) is shown early in the C _m trace. Histamine (100 μm) was added by superfusion as indicated. Increase in C _m (open arrow) on Ca²⁺ release from internal stores (filled arrow) was rarely observed (3 of 23 noninfected and infected cells). E, Mean ± SEM percentage change in exocytotic efficiency induced by histamine in noninfected cells (n = 8) compared with adenoviral infected cells expressing EGFP (n = 11). **p < 0.01.

Figure 5.

Histamine-induced potentiation of exocytosis is abolished in cells expressing DAG-insensitive Munc13-1^H567K. A, Sample I _Ca (top) and corresponding ΔC _m (bottom) evoked in an adenovirus-infected chromaffin cell expressing Munc13-1–EGFP before (ctrl) and during superfusion with histamine (100 μm). B, Sample I _Ca (top) and corresponding ΔC _m (bottom) evoked in an adenovirus-infected chromaffin cell expressing Munc13-1^H567K–EGFP before (ctrl) and during superfusion with histamine. C, Summary of effects of histamine on exocytotic efficiency normalized to (ctrl) for culture-matched, noninfected cells (n = 11), Munc13-1–EGFP-overexpressing (n = 10), and Munc13-1^H567K–EGFP-expressing (n = 6) cells. D, Corresponding summary of effects of histamine on normalized Ca²⁺ entry for the same groups of cells. Error bars indicate SEM. **p < 0.01; *p < 0.05.