Interaction Of Munc18 And Syntaxin In The Regulation Of Insulin Secretion In Pancreatic β Cells

Insulin is one of the most important versatile hormones, and its functions includeregulation of glycemia homeostasis, enhancement of anabolism, control of cell divisionand differentiation, and modulation of cell growth and development. Insulin is stored inlarge dense core vesicles and released by exocytosis, a multistage process involvingt-ransport of vesicles to the membrane, their docking, priming and final fusion with theplasma membrane. Syntaxin 1A (Stx1A) and Munc18a play essential roles in vesiculartrafficking and exocytosis. The molecular mechanism and the functional roles of theirinteraction remain to be explored. In the present study, we have studied the intracellularlocalization and interaction of Stx1A and Munc18a in insulin secreting INS-1 cells as wellas non-secretary CHO cells by confocal and evanescent field fluorescence imaging. Wefound that Munc18a colocalized with clusters of Stx1A and its open-form mutant, but notthe mutant lacking Habc domain, at the plasma membrane in live INS-1 cells, suggestingthe interaction of Munc18a with the Habc domain of Stx1A is necessary for thetranslocation of Munc18a to the plasma membrane. In CHO cells, where no endogenousStx1A is reported, Stx1A failed to localize to the plasma membrane even in the presenceof coexpressed Munc18a, suggesting Munc18a is not required for the transportation ofStx1A to the plasma membrane. On the other hand, Munc18a, which displayed a diffusedcytoplasmic localization alone in INS-1 cells, was translocated to the plasma membranewhen coexpressed with Stx1A, suggesting that the trafficking of Stx1A drives Munc18a tothe functional sites. Interestingly, Stx1A-L165A/E166A (Stx1A-DM), an openconfiguration of Stx1A that does not interact with Munc18a in a pull down assay, alsoexisted in clusters on the plasma membrane and Munc18a was concentrated on the samemicrodomains of Stx1A-DM, suggesting either Munc18a is recruited to the microdomainsthrough binding to the open-form Stx1A-DM. in vivo FRET measurement demonstratedthe interaction between Munc18a and Stx1A, whereas no significant FRET signal was IIIobserved for Munc18a and the open-form Stx1A mutant despite their colocalization,implying the different ways of interaction between Stx1A and Stx1A-DM with Munc18a. By overexpression of Munc18a in primary cultured pancreatic β cells, we have shownthat in response to the first flash, the slow burst component was inhibited as well as thesustained component of exocytosis, whereas the fast burst component remains unaltered.This effect is more obvious when the responses to the second flash were compared, wherethe recovery of both the fast burst and the slow burst component was blocked. The resultsfavor a role for Munc18a to block the formation of trans-SNARE complexes between thevesicle and the plasma membrane by stabilizing Syntaxin in closed configuration, henceinhibiting the recruitment of vesicle to the release-ready pools. Additionally, we havefound that overexpression of neither the wild type Stx1A nor the open-form Stx1A affectsexocytosis, suggesting the availability of Stx1A in its open form is not a rate limiting stepin insulin secretion.