The slower kinetics of insulin release from pancreatic islet beta cells, as compared with other regulated secretory processes such as chromaffin granule secretion, can in part be explained by the small number of the insulin granules that are docked to the plasma membrane and readily releasable. In type-2 diabetes, the kinetics of insulin secretion become grossly distorted, and, to therapeutically correct this, it is imperative to elucidate the mechanisms that regulate priming and secretion of insulin secretory granules. Munc13-1, a synaptic protein that regulates SNARE complex assembly, is the major protein determining the priming of synaptic vesicles. Here, we demonstrate the presence of Munc13-1 in human, rat, and mouse pancreatic islet beta cells. Expression of Munc13-1, along with its cognate partners, syntaxin 1a and Munc18a, is reduced in the pancreatic islets of type-2 diabetes non-obese Goto-Kakizaki and obese Zucker fa/fa rats. In insulinoma cells, overexpressed Munc13-1-enhanced green fluorescent protein is translocated to the plasma membrane in a temperature-dependent manner. This, in turn, greatly amplifies insulin exocytosis as determined by patch clamp capacitance measurements and radioimmunoassay of the insulin released. The potentiation of exocytosis by Munc13-1 is dependent on endogenously produced diacylglycerol acting on the overexpressed Munc13-1 because it is blocked by a phospholipase C inhibitor (U73122) and abrogated when the diacylglycerol binding-deficient Munc13-1 H567K mutant is expressed instead of the wild type protein. Our data demonstrate that Munc13-mediated vesicle priming is not restricted to neurotransmitter release but is also functional in insulin secretion, where it is subject to regulation by the diacylglycerol second messenger pathway. In view of our findings, Munc13-1 is a potential drug target for therapeutic optimization of insulin secretion in diabetes.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane-localized chloride channel. Some mutations in CFTR, including one which affects most patients (delta F508-CFTR), prevent CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized. To examine whether normal and mutant CFTRs function as chloride channels when they reside in the ER, the patch clamp technique was used to measure currents in the outer membrane of nuclei isolated from mammalian cells expressing CFTR. Both delta F508-CFTR as well as CFTR were revealed to function as cAMP-regulated chloride channels in native ER membrane. These results represent the first demonstrations of functional activity of CFTR in the biosynthetic pathway and suggest that conformational changes in the mutant protein, although recognized by ER-retention mechanisms, do not necessarily affect CFTR chloride channel properties, which may have implications for pathophysiology and therapeutic interventions in cystic fibrosis.

Cystic fibrosis (CF) is characterized by abnormal regulation of epithelial ion and fluid transport due to mutations in the CF transmembrane conductance regulator (CFTR), an apical membrane-localized Cl ؊ channel, that usually prevent it from exiting the endoplasmic reticulum. Defective or absent CFTR in the epithelium is believed to disrupt fluid balance in human airways and thereby contribute to chronic respiratory inflammation. Patch-clamp of the plasma membrane and outer membrane of the nuclear envelope of nuclei isolated from CFTR-expressing Chinese hamster ovary cells revealed that CFTR is associated with a regulated ATP channel in both membrane compartments. CFTR expression was also shown to be associated with permeability to another adenine nucleotide, adenosine 3-phosphate 5-phosphosulfate, the universal sulfate donor in cells. These results may provide a link between the ion channel function of CFTR and abnormal glycoprotein processing observed in CF.

Studies of rat aorta revealed that cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum Ca2+ pump, released endothelium-derived relaxing factor (EDRF) and relaxed the muscle. We have used CPA to elucidate how this inhibitor of Ca2+ uptake into internal stores affects K+ channels and Ca2+ entrance in cultured bovine pulmonary endothelial cells using patch-clamp techniques. CPA increased a Ca(2+)-dependent outward K+ current for many minutes, presumably as a consequence of the unbalanced leakage of Ca2+ from internal stores and Ca2+ entrance across the cell membrane. An expected consequence of this activation of the outward current change is hyperpolarization of the cell membrane and increased driving force for Ca2+ entry. CPA activated the influx of extracellular Ca2+ through nonselective cation channels. Ca2+ influx through nonselective cation channels could help maintain intracellular Ca2+ concentration elevation and EDRF release. CPA also reduced the inwardly rectifying K+ current. Inositol 1,4,5-trisphosphate (IP3) in the patch pipette also produced an increase in outward K+ currents, which were Ca2+ dependent. After depletion of Ca2+ internal stores by CPA, the response to IP3 was abolished. Heparin in the patch pipette reduced the increase in outward currents induced by bradykinin, an agonist known to raise IP3 and to release Ca2+, but did not prevent CPA-induced increases in outward current. Thus CPA acts to elevate Ca(2+)-activated currents in endothelial cells by a mechanism independent of IP3-induced release, and this may lead to EDRF release both directly and as a consequence of Ca2+ entry through nonselective cation channels driven by an increased electrical gradient for Ca2+.

The R domain of cystic fibrosis transmembrane conductance regulator (CFTR) connects the two halves of the protein, each of which possess a transmembranespanning domain and a nucleotide binding domain. Phosphorylation of serine residues, which reside mostly within the C-terminal two-thirds of the R domain, is required for nucleotide-dependent activation of CFTR chloride channel activity. The N terminus of the R domain is also likely to be important in CFTR function, since this region is highly conserved among CFTRs of different species and exhibits sequence similarity with the "linker region" of the related protein, P-glycoprotein. To date, however, the role of this region in CFTR channel function remains unknown. In this paper, we report the effects of five disease-causing mutations within the N terminus of the CFTR-R domain. All five mutants exhibit defective protein processing in mammalian HEK-293 cells, suggesting that they are mislocalized and fail to reach the cell surface. However, in the Xenopus oocyte, three mutants reached the plasma membrane. One of these mutants, L619S, exhibits no detectable function, whereas the other two, D614G and I618T, exhibit partial activity as chloride channels. Single channel analysis of these latter two mutants revealed that they possess defective rates of channel opening, consistent with the hypothesis that the N terminus of the R domain participates in ATP-dependent channel gating. These findings support recent structural models that include this region within extended boundaries of the first nucleotide binding domain.