Molecular analysis of TRPV6 protein function

Our current knowledge on the transient receptor potential (TRP) superfamily suggests some of these proteins as essential elements of cation channels affecting capacitive Ca2+ entry (CCE). TRPV6 proteins have recently emerged as Ca2+-selective ion channels, the expression of which has been detected in various secretory cells. This project aims at the molecular analysis of TRPV6 protein function in calcium influx pathways of mast cells and prostate cancer cells. For this, several techniques such as electrophysiology, fluorescence microscopy, molecular biology and in vitro biochemical studies will be employed. The contribution of TRPV6 proteins to CCE in mast cells and prostate cancer cells will be evaluated by TRPV6 antisense and RNAi strategy as well as by the use of dominant negative TRPV6 mutants. Among these mutants, the amino-terminus of TRPV6 is expected to function as dominant negative, as it might interfere with the formation or/and function of tetrameric Ca2+ channels. To resolve the mechanism(s) of this dominant negative effect, the intermolecular interactions mediating TRPV6 self-assembly will be studied by using TRPV6 amino-terminal fragments and mutants. Further potential interactions of TRPV6 with e.g. the IP 3 receptor, phospholipase C- and NHERF will be investigated for their involvement in the regulation of TRPV6 activity. As TRPV6 activation is inhibited by the compound 2-APB, a derivative of this compound will be used to additionally identify proteins affecting TRPV6 activity in CCE. The anticipated gain in the knowledge on the regulation of TRPV6 channels will lead to a molecular understanding of Ca2+ responses in mast cells and prostate cancer cells thereby increasing the possibilities for therapeutic manipulation.