Interaction between TRPC1 and TRPCV6

The transient receptor potential (TRP) proteins assemble to form cation-permeable channels. Beside predominant homomeric TRP channels, heteromeric TRP channels have been identified, mainly within homological subfamilies. The only TRP protein which is known to interact with members of other TRP subfamilies is TRPC1 of the canonical TRPs. This project focuses on the interaction of TRPC1 with TRPV6, a protein from the vanilloid-type TRP subfamiliy. In a combined approach several techniques including patch-clamp, fluorescence microscopy as well as molecular recognition force microscopy will be complemented by molecular biology and biochemical studies to resolve TRPC1/TRPV6 interaction both in vitro and at the functional level in living cells. In the latter, the cellular/molecular consequences of this interaction will be evaluated to gain insight how TRPC1 modulates TRPV6 activity. We will investigate whether novel heteromeric TRPC1/TRPV6 channels will be formed as a direct consequence of this interaction or whether TRPC1 more indirectly affects the formation of functional TRPV6 channels. Moreover the molecular determinants mediating heteromeric interaction of these channels will be examined, and a quantitative analysis of their affinities correlated with molecular forces will enable to determine key domains for TRPC1 as well as TRPV6 homomeric and TRPC1/TRPV6 heteromeric interactions. The N- termini of both channels contain candidate structures for these interactions such as coiled-coil domains or ankyrin- like repeats that are known to be important for protein-protein interactions. It will be particularly interesting to resolve whether molecular determinants for TRPC1/TRPV6 heteromeric interactions are distinct to those mediating homomeric interactions as this would allow differential modulation of homo- and heteromeric channels by corresponding dominant negative constructs. A profound understanding of the novel interaction of TRPV6 and TRPC1 will then provide the basis to molecularly resolve store-operated Ca2+ channels (SOC) in prostate carcionoma cells (LNCaP) as both TRPC1 and TRPV6 proteins are here in the focus as molecular components of mediating SOC.

This project initially focused at the interaction of TRPC1 with TRPV6, proteins from the canonical and vanilloid- type TRP subfamiliy. A potential interaction of these two proteins has been proposed from store-operated currents (SOC) in prostate cancer LNCaP cells, which are inhibited by dominant negative species of either TRP channel. The SOC current in LNCaP cells additionally involved two proteins, STIM1 and ORAI1 that have been recently identified as key players for the best characterized SOC, the Ca2+release activated Ca2+ current (CRAC) in immune cells. Therefore, focus of the project was extended onto the investigation of the STIM1-ORAI1 system in as that a characterization of the coupling of these two proteins might be relevant for the understanding of SOC in LNCaP cells together with the proposed modulation of TRPV6 activity by TRPC1. The latter was initially characterized as an inhibitory effect of TRPC1 on TRPV6 plasma membrane expression levels based on a dominant negative TRPC1 N-terminus. Investigation of the coupling of STIM1 to Orai similarly utilized a combined approach by several techniques including patch-clamp and dynamic Förster Resonance Energy Transfer (FRET) microscopy, complemented by molecular biology and biochemical studies to resolve STIM1/ORAI1 interaction both in vitro and at the functional level in living cells. In this project we further demonstrated by FRET microscopy a dynamic coupling of STIM1 and ORAI1 that culminated in the activation of Ca2+ entry. FRET imaging of living cells provided insight in the time-dependence of crucial events of this signalling pathway comprising Ca2+ store depletion, STIM1 multimerization and STIM1-ORAI1 interaction. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The dynamic interaction occurred via the C-terminus of ORAI1 that includes a putative coiled-coil domain structure. Within STIM1 cytosolic portion, we identified an ORAI1 activating small fragment (OASF) comprising two coiled-coil domains and additional 50 amino acids that exhibited enhanced interaction with ORAI1 resulting in three-fold increased Ca2+ currents. In aggregate, we have identified two cytosolic key regions within STIM1/ORAI1 mediating their interaction via coiled-coil regions in respective C-termini. The understanding of the coupling mechanism of STIM1 and ORAI1 represents a promising basis for the development of drugs that modulate SOC.