Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release

Abstract

Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a ‘conventional’ PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP.Brain function depends on the rapid transfer of information from one brain cell to the next at junctions known as synapses. Small packages called vesicles play an important role in this process. The arrival of an electrical action potential at the nerve terminal of the first cell causes some vesicles in the cell to fuse with the cell membrane, and this leads to the neurotransmitters inside the vesicles being released into the synapse. The neurotransmitters then bind to receptors on the second cell, which leads to an electrical signal in the second cell. A protein called Munc18-1 has a central role in the fusion of the vesicle at the cell membrane.The strength of a synapse—that is, how easily the first brain cell can impact the electrical behaviour of the second—can change, and this ‘synaptic plasticity’ is thought to underlie learning and memory. Long-term changes in synaptic strength require additional receptors to be inserted into the membrane of the second cell. However, synapses can also be temporarily strengthened: the arrival of a burst of action potentials—a tetanus—causes some synapses to increase the amount of neurotransmitter they release in response to any subsequent, single, action potential.This temporary increase in synaptic strength, which is known as post-tetanic potentiation, requires an enzyme called protein kinase C; the role of this enzyme is to phosphorylate specific target proteins (i.e., to add phosphate groups to them). Now, Genç et al. have genetically modified a mouse synapse in vivo and shown that protein kinase C brings about post-tetanic potentiation by phosphorylating Munc18-1. Furthermore, pharmacological experiments show that proteins called phosphatases, which de-phosphorylate proteins, normally terminate the post-tetanic potentiation after about one minute. Taken together, the study identifies a target protein which is phosphorylated by protein kinase C during post-tetanic potentiation. The study also suggests that in addition to its fundamental role in vesicle fusion, the phosphorylation state of Munc18-1 can change the probability of vesicle fusion in a more subtle way, thereby contributing to synaptic plasticity.