All-Optical Analysis of TRPC3/6 Signalling in Mast Cells

Abstract

Transient receptor potential canonical channels TRPC3/6 are expressed in various cell types of the immune system. These Ca2+ permeable channels are typically activated downstream of the phospholipase C (PLC) pathway, but their role in immune cell Ca2+ handling is still elusive. We set out to explore the impact of TRPC3 and TRPC6 function in RBL mast cells at the level of local, sub-plasmalemmal and global cellular Ca2+ signalling, independently of PLC activity by using an all-optical approach. C-terminal fusions of TRPC3 and TRPC6 with R-GECO1.0 were generated to record Ca2+ changes in the vicinity of the TRPC channels. A new photochromic benzimidazole (OptoBI-1) was used to generate temporally precise pattern of channel activity in the absence of PLC signalling. When expressed in HEK293 cells, TRPC3 or TRPC6 activation generated both local and global Ca2+ rises, while TRPC channels expressed in RBL mast cells produced large local Ca2+ elevation that was associated with barely detectable global Ca2+ rises. Interestingly, disruption of lipid accommodation within the channels' pore domain (mutation in the lipid gating window) only moderately modified kinetics of local Ca2+ rises but completely restored the associated global cytosolic Ca2+ response. Our all-optical analysis of TRPC signal transduction in RBL mast cells suggest that minor changes in TRPC lipid coordination and current kinetics are of crucial impact on overall TRPC signalling features in immune cells.