Many key proteins involved in membrane targeting and synaptic vesicle neurotransmitter release have been identified and a fundamental set of interactions defined and placed in a model termed the SNARE hypothesis. However, despite recent rapid progress in identifying molecular components of the membrane targeting and fusion machine, regulatory influences facilitating or inhibiting the protein interactions or the sequence of interactions remain poorly defined. It is the long range goal of the proposed research to identify and understand the regulatory mechanism(s) which govern SNARE protein interactions and, thereby, regulate neurotransmitter and neurohormone release and synaptic plasticity. Preliminary molecular/biochemical studies combined with functional studies monitoring membrane capacitance changes, as a measure of exo-endocytotic activity under whole cell patch clamp, have indicated that proteins of the Sec1 family serve an important regulatory control function. Experiments proposed will test the hypothesis that nSec1 protein (nSec1p) regulates neurosecretion via a specific regulated interaction with syntaxins and that this interaction enhances secretory granule docking. The proposed experiments will utilize nerve endings of the hypothalamo-neurohypophysial system, which possess unique anatomical and electrophysiological advantages allowing resolution of the molecular events of the secretory process at nerve endings to be studied in greater detail than at any other nerve endings. The investigators will utilize a combination of molecular, biochemical, and patch clamp techniques to characterize the regulation of nSec1 protein interactions and to analyze functional effects on Ca2+ currents and on the amplitude and kinetics of secretion with msec resolution at individual nerve endings.
The specific aims are: 1) to determine the sites of phosphorylation on nSec1p and examine regulation of protein interactions by specific kinase activity and depolarizing stimuli, 2) to determine the mechanism by which nSec1p regulates secretory granule exocytosis and elucidate the effects of phosphorylation state on secretion, 3) to determine if nSec1p regulation of exocytosis utilizes the molecular mechanisms and machinery proposed by the SNARE hypothesis, and 4) to determine if nSec1p interacts with and activates cdk5, whether this interaction is regulated by nSec1p phosphorylation, and if there are functional consequences on secretion. In summary, an understanding of the mechanisms by which nSec1p regulates SNARE protein interactions may be essential to full understanding of both short and long-term processes of synaptic plasticity and memory.
