Ca2+ controls cellular processes, ranging from secretion, generation of mediators and gene transcription to cell proliferation. Ca2+ entry can be initiated via a lipid messenger-dependent receptor-operated or an intracellular Ca2+ store filling state-dependent, store-operated entry pathway. Aside voltage-gated channels, TRPCs and Orai-channels are key proteins for these processes.Aberrancies in Ca2+ entry can lead to nephritic diseases, progressing malfunctions of motor-coordination, cardiac pathologies or immune deficiency. TRPC3 represents a prototypical TRPC cation channel that is involved in ROCE and SOCE. Understanding its function and crosstalk with Orai1, the pore-forming protein of SOCE, is expected to lead to novel strategies for the treatment of pathological conditions based on dysfunction of cellular Ca2+ handling.Combining molecular modelling and mutagenesis of TRPC3 led to the identification of a single AA in the TRPC3 putative pore region, which is essential for Ca2+ permeation. TRPC3E630Q reduced divalent permeability of the channel by more than two orders of magnitude and TRPC3E630K was found to eliminate cation permeability completely. The mutations were found to act dominant-negatively on endogenous TRPCs.Pharmacological modulation of TRPC3 and Orai1 was characterized for several pyrazoles. A compound designated Pyr6, potently and selectively blocked Orai1-mediated SOCE while a newly synthesised structure, Pyr10, displayed selectivity for block of TRPC3. Using these tools, evidence was obtained for a signalplex, including TRPC3 either as a scaffold protein or as signalling component essential for Ca2+ entry and targeting of Ca2+ signalling components into regulatory membrane and transcriptionally active microdomains of RBL-2H3 and HL-1 cells. The identified scaffold function of TRPC3 was dynamically regulated by reversible PKC-phosphorylation.These results shed light on molecular function and pathophysiological role of TRPC3.