Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.Protein phosphorylation has been long known as an important mechanism for the regulation of exocytosis although, with only a few exceptions such as the synapsins (1), the targets for regulation by phosphorylation in vivo are unknown. Treatment with phorbol esters modifies regulated exocytosis in many different neuronal and non-neuronal (2, 3) cell types leading to increased vesicle recruitment into the ready releasable pool (4 -6), acceleration of fusion pore expansion (7), or changes in the kinetics of exocytosis (8,9). PKC also has a key role in synaptic plasticity (10). The effects of phorbol ester were originally attributed to activation of PKC 1 although the PKC substrates responsible had not been identified, and it is not known if the same target regulates all of the parameters modified by phorbol esters. The SNARE proteins, syntaxin 1, SNAP-25, and VAMP play key roles in exocytosis (11-13), and formation of the SNARE complex has been suggested to be a driving force for membrane fusion (14). The syntaxin-binding protein Munc18a (15) (29) and synaptotagmin I (25). In no case has the functional consequences of these phosphorylation events for exocytosis been established. Indeed, the phosphorylation of SNAP-25 by PKC in PC12 cells lagged well behind the effects of phorbol ester on the extent of exocytosis (29). In that study, it was also shown that the phorbol ester effects had both a PKC-dependent and a PKC-independent component. The synaptic protein Munc13 has been identified as an alternative phorbol ester-binding protein (30, 31), and recently it has been suggested that the effects of phorbol ester on synaptic transmission are mediat...

Protein SUMOylation is a critically important posttranslational protein modification that participates in nearly all aspects of cellular physiology. In the nearly 20 years since its discovery, SUMOylation has emerged as a major regulator of nuclear function, and more recently, it has become clear that SUMOylation has key roles in the regulation of protein trafficking and function outside of the nucleus. In neurons, SUMOylation participates in cellular processes ranging from neuronal differentiation and control of synapse formation to regulation of synaptic transmission and cell survival. It is a highly dynamic and usually transient modification that enhances or hinders interactions between proteins, and its consequences are extremely diverse. Hundreds of different proteins are SUMO substrates, and dysfunction of protein SUMOylation is implicated in a many different diseases. Here we briefly outline core aspects of the SUMO system and provide a detailed overview of the current understanding of the roles of SUMOylation in healthy and diseased neurons.

SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (K ATP ) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotidebinding domains of SUR results in an increase in K ATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.

The ATP‐sensitive potassium (KATP) channel couples glucose metabolism to insulin secretion in pancreatic β‐cells. It comprises regulatory sulfonylurea receptor 1 and pore‐forming Kir6.2 subunits. Binding and/or hydrolysis of Mg‐nucleotides at the nucleotide‐binding domains of sulfonylurea receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also compared the ATPase activity of sulfonylurea receptor 1 with that of the isolated nucleotide‐binding domains (fused to maltose‐binding protein to improve solubility). Electron microscopy showed that nucleotide‐binding domains purified as ring‐like complexes corresponding to ∼ 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol Pi·s−1·(nmol protein)−1] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea receptor 1, nucleotide‐binding domain 1, nucleotide‐binding domain 2 and a mixture of nucleotide‐binding domain 1 and nucleotide‐binding domain 2, respectively. Corresponding Km values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower Km than either of the isolated nucleotide‐binding domains, and a lower maximal turnover rate than nucleotide‐binding domain 2. Similar results were found with GTP, but the Km values were lower. Mutation of the Walker A lysine in nucleotide‐binding domain 1 (K719A) or nucleotide‐binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (Ki 16 µm), but not MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of the nucleotide‐binding domains. These data demonstrate that the ATPase activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide‐binding domains, suggesting that the transmembrane domains may influence the activity of the protein.