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David Stevens:
Modulation of neurotransmitter and hormone release

Research

Neurotransmitters are released under tightly controlled conditions in response to appropriate stimuli. Release is the result of exocytosis induce d by an increase in intracellular calcium. Transmitter-filled vesicles fuse with the cell membrane, setting their content free. Fusion is controlled by SNARE complexes consisting of the proteins SNAP-25, Syntaxin and synaptobrevin (Jahn and Fasshauer, 2012). These complexes generate the force which drives the fusion of vesicles with the plasma membrane. The assembly and function of SNARE complexes are controlled by a number of proteins allowing a fine tuning of release.

Of particular note are the Munc18 family which control assembly of a vesicle acceptor complex which allows docking of vesicles at the plasma membrane, synaptotagmins, which endow the SNARE complex with the ability to respond rapidly to changes in intracellular calcium, and the priming factors Munc13 and CAPS which are needed to render the SNARE complex fusion competent. Once primed, the vesicles await an adequate calcium stimulus which induces fusion.

We are primarily interested in the function of the priming factors Munc13 and CAPS. We use mouse chromaffin cells in primary culture (Fig. 1E) as a model cell for calcium-stimulated exocytosis. We use electrophysiological methods, in particular capacitance measurements to monitor exocytosis and carbon fiber amperometry to detect catecholamine release to determine the amounts and rates of the release of the catecholamines from these cells. In addition we use high resolution imaging, electron microscopy, biochemical and molecular biological methods to study the priming process.

We have carried out structure-functions studies on Munc13-1 (Stevens et al., 2005) and studies of CAPS function in exocytosis (Liu et al., 2008, 2010; Speidel et al., 2005). Both molecules are able to facilitate catecholamine release by increasing vesicle priming. The domain structure of CAPS1 and Munc13-1 are shown below. The figure is from our review of the CAPS literature (Stevens and Rettig, 2009). We are currently extending these studies. 

Figure 1: CAPS function in exocytosis

A. The domain structure of CAPS and that of the priming factor Munc13-1. CAPS contains a N-terminal dynactin 1 binding domain (DBD). The central portion of CAPS contains two lipid interacting domains, the C2 domain whose function is unclear, and the PH domain which is necessary for an interaction with PIP2 rich areas in the inner leaflet of the plasma membrane. Distal to these domains are a Munc-13 homology domain including the syntaxin interaction domain (SID) required for priming in SVs and a C-terminal domain required for association with DCVs. Thus CAPS contains a MHD domain which in Munc13 is part of the minimal structure required to carry out priming (MUN domain).

Chromaffin cell

B, C. Current models suggest the primed vesicle can exist in two states, a slowly releasable and a rapidly releasable state. Priming is likely a sequential process with docked vesicles, which are recruited from a depot pool, being primed to the slowly releasable pool (SRP) and then proceeding to the rapidly releasable pool (RRP).

D. High time resolution experiments in mouse chromaffin cells indicate that CAPS acts in conversion of SRP vesicles to rapidly releasable vesicles since the RRP and sustained release observed in wild type mice (wt) are strongly reduced in CAPS knock-out mice (dko) and this effect can be rescued by ectopic CAPS1 expression (rescue).

E. Electron microgragh of a mouse chromaffin cell. Arrows indicate vesicles docked at the membrane. The scale bar indicates 500 nm.

References

Jahn R, Fasshauer D (2012). Molecular machines governing exocytosis of synaptic vesicles. Nature 490, 201-207.

Liu Y, Schirra C, Stevens DR, Matti U, Speidel D, Hof D, Bruns D, Brose N, Rettig J (2008). CAPS facilitates filling of the rapidly releasable pool of large dense-core vesicles. J Neurosci 28, 5594-5601.

Liu Y, Schirra C, Edelmann L, Matti U, Rhee J, Hof D, Bruns D, Brose N, Rieger H, Stevens DR, et al. (2010). Two distinct secretory vesicle-priming steps in adrenal chromaffin cells. J Cell Biol 190, 1067-1077.

Speidel D, Bruederle CE, Enk C, Voets T, Varoqueaux F, Reim K, Becherer U, Fornai F, Ruggieri S, Holighaus Y, et al. (2005). CAPS1 regulates catecholamine loading of large dense-core vesicles. Neuron 46, 75-88.

Stevens DR, Rettig J (2009). The Ca(2+)-dependent activator protein for secretion CAPS: do I dock or do I prime? Mol Neurobiol 39, 62-72.

Stevens DR, Wu ZX, Matti U, Junge, HJ, Schirra C, Becherer U, Wojcik SM, Brose N, Rettig J (2005). Identification of the minimal protein domain required for priming activity of Munc13-1. Curr Biol 15, 2243-2248.