The Ca²⁺-dependent non-selective cation channel TRPC5 is a membrane protein with a prominent role in providing a route for Ca²⁺ entry into excitable cells. TRPC5 has been reported to regulate neurite extension and growth-cone morphology in hippocampal neurons (Greka et al, 2003). While the nature of the moieties initiating channel gating remain elusive, it is apparent that TRPC5 gating is complex and subject to regulation by many signals arising close to the plasma membrane (Strübing et al, 2001). Here we examine whether NCS-1 (Burgoyne & Weiss, 2001) plays a role in TRPC5 function in HEK293 cells, stably expressing tetracycline-regulated transcription of human TRPC5 (hTRPC5). [Ca²⁺]i was monitored using Fura-PE3. Ca²⁺ signals from other cation channels were inhibited by 10 µM Gd³⁺.The responses of hTRPC5 to a variety of activating signals were assessed in cells expressing either dominant-negative NCS-1 (E120Q; NCS1-DN) or dominant-negative calmodulin (no intact EF-hands; CaM DN). Data are presented as mean ± S.E.M. with n indicating the number of cells followed by the number of experiments in parentheses. Statistical significance was determined using an unpaired 2-tailed Student’s t test. After depletion of intracellular Ca²⁺ stores with 1 µM thapsigargin, store- and receptor- operated hTRPC5 activity was assessed. Muscarinic receptors were activated by 100 µM carbachol. Gd³⁺ (100 µM) was used in separate experiments to activate hTRPC5 directly. All modes of hTRPC5 activity were significantly inhibited by NCS-1 DN (p<0.001; n = 123 (6-11)). By contrast, CaM DN expression resulted in no inhibition of hTRPC5 activity (n = 49 (3)). Mouse TRPC5 is known to be regulated by phospholipase C activity and we suggest a similar link for hTRPC5 because an inhibitor of phospholipase C, U73122 (10 µM), strongly inhibited the activity of hTRPC5. It seems plausible that NCS-1 DN affected hTRPC5 because it displaced endogenous NCS-1 from one of its established protein partners, PI4 kinase (Zhao et al, 2001) thereby reducing PIP2 levels. We tested this idea by inhibiting PI4 kinase with wortmannin (10 µM; 30 min). Unexpectedly, we found that store-operated TRPC5 activity was unaffected after wortmannin treatment (n = 303 (2)). Therefore it seems unlikely that NCS-1 DN affects TRPC5 via PI4 kinase. We suggest NCS-1 plays a role in the function of hTRPC5, possibly mediating the stimulatory effect of Ca²⁺ on its channel activity.