Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, C88

Oral Communications

Contribution of TRPC1 to store-mediated regulation of Ca2+-sensitive adenylyl cyclases

D. Willoughby1, H. Ong2, I. S. Ambudkar2, D. M. Cooper1

1. Pharmacology, University of Cambridge, Cambridge, United Kingdom. 2. Secretory Physiology Section, NIDCR, National Institutes of Health, Bethesda, Maryland, United States.

Store-operated Ca2+ entry (SOCE) is mediated via specific plasma membrane channels in response to the depletion of endoplasmic reticulum (ER) Ca2+ stores. Two key components of store-operated Ca2+ channels (SOCCs) are known. Stromal interacting molecule 1 (STIM1) senses ER Ca2+ store content and translocates to the plasma membrane upon store depletion where it associates with a second protein, Orai1, which is thought to act as a pore-forming component of SOCCs (1). Members of the canonical transient receptor potential (TRPC) family of Ca2+ channels are also putative candidates for SOCCs, with evidence of interaction between TRPCs, Orai1 and STIM1 (2). Recent work by our laboratory has revealed that SOCE reconstituted by co-expression of STIM1 and Orai1 in HEK293 cells can activate Ca2+-stimulated adenylyl cyclase type 8 (AC8) which responds selectively to SOCE over other types of Ca2+ rise (including IP3-mediated Ca2+ release and arachidonate- or ionophore-mediated Ca2+ entry) (3). Here, we address whether TRPC1, a contentious component of SOCCs alongside Orai1 and STIM1, plays any part in SOCE-mediated regulation of Ca2+ sensitive ACs. Using a genetically-encoded Ca2+ sensor, GCaMP2, we examined the effects of TRPC1 expression and selective knockdown on SOCE signals in HEK293 cells stably expressing AC8 (HEK-AC8) and human submandibular gland (HSG) cells endogenously expressing Ca2+-inhibitable AC6. Co-expression of TRPC1 with STIM1 and Orai1 potentiated SOCE to 182±7% and 306±12% of controls in HEK-AC8 cells and HSG cells respectively. This was significantly greater than the effects of co-expressing STIM1 and Orai1 without TRPC1 (158±8% of control in HEK-AC8 cells and 217±13% in HSG cells) (p<0.05, n=37-68). In contrast, expression of shRNA directed against human TRPC1 reduced Ca2+ entry by 24±6% in HEK-AC8 cells and 36±11% in HSG cells when compared to controls (p<0.05, n=23-62). Parallel experimental conditions assessed the effects of TRPC1-dependent Ca2+ entry on cAMP production using a real-time FRET-based cAMP sensor, Epac2-camps (4). Expression of TRPC1 alongside STIM1 and Orai1 in HEK-AC8 cells increased Ca2+-dependent AC8 activity to 27±8% above that seen following expression of STIM1 and Orai1 alone (p<0.05, n=18-21). Knockdown of TRPC1 levels reduced SOCE-stimulated AC8 activity by 50±14% compared to controls in HEK-AC8 cells (p<0.01, n=16-19). In HSG cells treated with shRNA for TRPC1, SOCE-mediated inhibition of AC6 activity was reduced to 51±11% of controls (p<0.01, n=12-16). All experiments were performed at RT using an EMCCD-camera imaging system. Our findings suggest that TRPC1 is an integral component of SOCCs in HEK293 and HSG cells where it associates alongside STIM1 and Orai1 to mediate Ca2+ fluxes within AC8 and AC6 cellular microdomains to regulate cAMP production.

Where applicable, experiments conform with Society ethical requirements