Activity-Dependent Fusion Pore Expansion Regulated by a Calcineurin-Dependent Dynamin-Syndapin Pathway in Mouse Adrenal Chromaffin Cells

Abstract

Neuroendocrine chromaffin cells selectively secrete a variety of transmitter molecules into the circulation as a function of sympathetic activation. Activity-dependent release of transmitter species is controlled through regulation of the secretory fusion pore. Under sympathetic tone, basal synaptic excitation drives chromaffin cells to selectively secrete modest levels of catecholamine through a restricted secretory fusion pore. In contrast, elevated sympathetic activity, experienced under stress, results in fusion pore expansion to evoke maximal catecholamine release and to facilitate release of co-packaged peptide transmitters. Therefore, fusion pore expansion is a key control point for the activation of the sympatho-adrenal stress response. Despite the physiological importance of this process, the molecular mechanism by which it is regulated remains unclear. Here we employ fluorescence imaging with electrophysiological, and electrochemical-based approaches to investigate the role of dynamin I in the regulation of activity-mediated fusion pore expansion in mouse adrenal chromaffin cells. We show that under elevated stimulation, dynamin I is dephosphorylated at Ser-774 by calcineurin. We also demonstrate that disruption of dynamin I-syndapin binding, an association regulated by calcineurin-dependent dynamin dephosphorylation, limits fusion pore expansion. Lastly, we show that perturbation of N-WASP function (a syndapin substrate) limits activity-mediated fusion pore expansion. Our results suggest that fusion pore expansion is regulated by a calcineurin-dependent dephosphorylation of dynamin I. Dephosphorylated dynamin I acts via a syndapin/N-WASP signaling cascade to mediate pore expansion.

Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells

“…Our data show that, unlike neurons, where dynamin-syndapin 1 regulates synaptic vesicle function (41), neuroendocrine cells utilize a dynamin-syndapin 3 system for modulation of fusion pore expansion. We previously demonstrated that dynamin 1-syndapin interaction and neural Wiskott-Aldrich syndrome protein (N-WASP) activation play a critical role in mediating fusion pore behavior in neuroendocrine cell secretion (57). The syndapin 1 P437L, syndapin 2 P478L, and syndapin 3 P415L mutants used in the current study have been shown to lose binding affinity to PRD-containing proteins including dynamin 1, synaptojanin 1, and N-WASP (36,37,45).…”

“…However, if the cell is under heightened stimulation, as under the acute sympathoadrenal stress reflex, elevated Ca 2ϩ initiates calmodulin-activated calcineurin to dephosphorylate dynamin 1 at the PRD. This, in turn, reveals a binding site for the complementary SH3 domain of syndapin (57). This dephosphorylation step initiates the activation of a signaling cascade that ultimately leads to the regulated myosin-depen- value and also reflects a right-shifted dependent staining pattern (bottom middle and bottom right).…”

“…Transfected cells were identified by EGFP fluorescence, patchclamped, and stimulated with a train of APe delivered at 15 Hz. This stimulation paradigm has been shown to drive active expansion of the exocytic fusion pore to a high-conductance state (29) that is mediated by a dynamin 1-syndapin interaction (57). Evoked catecholamine release was simultaneously measured by electrochemical amperometry at the single-granule level (15,66).…”

“…Thus activity-regulated expansion of the secretory fusion pore has been proposed as a key mechanistic component of the sympathoadrenal stress reflex, increasing catecholamine quantal size and facilitating peptide hormone secretion from the dense granule core (22,27,50). We previously showed that fusion pore expansion is regulated by a calcineurin-activated dynamin 1-syndapin-dependent process (57). Syndapin is an accessory protein that regulates several endocytic processes, including receptor-mediated internalization and recycling of neuronal membrane during transmitter release (4,16,38,41,52).…”

“…Syndapin 3, the least characterized isoform (37), has been demonstrated to play a role in glucose transport (56) and transient receptor potential vanilloid 4 channel trafficking (17,18). We recently showed that interaction between dynamin 1 and an at-the-time unidentified member of the syndapin family contributes to regulated expansion of the fusion pore and facilitation of catecholamine release from neuroendocrine chromaffin cells (57). However, there is no available report regarding the expression profile of syndapin isoforms and their specificity of function in neuroendocrine chromaffin cells.…”