Redox microdomains and its implication in exocytosis in beta cells

Abstract

This project aims to better understand the effects of redox modulation on beta cells exocytosis and to identify the presence of redox domains in the vicinity of the plasma membrane in beta cells. Using small interference RNA (SiRNA) against p22PHOX, a NAD(P)H oxidase component, and the patch-clamp technique we will investigate the importance of this enzyme activity for the K+ membrane conductance via KATP channels, whole-cell and single channel conductance, and on the electrical activity induced by glucose. Additionally, using TIRF microscopy we aim to identify possible redox changes in regions of granule tethering to the membrane. This analysis will be possible by using membrane-targeted redox probes and granule-targeted probes based on fluorescent protein chimeras. Moreover, by combining voltage-clamp patch-clamp and TIRF or confocal microscopy techniques, redox changes in the membrane region in response to depolarization-evoked exocytosis will be investigated. The development of this project will contribute to a better understanding of redox domains in the vicinity of membrane and the possible implication of these domains in exocytosis. Additionally, this period in the Rorsman laboratory will allow a great experience in novel electrophyisiological and imaging techniques to be applied in our group in Brazil. (AU)