(A) Efficiency of TRPC1 knockdown using RNAi. PTH-C1 cells were transiently transfected with a scrambled siRNA (lane 1), siTRPC1 (lane 2), expression plasmid (positive control) of a long form of TRPC1α (lane 3), or a short form of TRPC1α (lane 4). TRPC1 was immunoprecipitated and immunoblotted with a TRPC1-specific monoclonal antibody (1F1). PTH-C1 cells express predominantly the long form of TRPC1α (indicated by an asterisk). (B) Changes in intracellular Ca²⁺ concentration (expressed as fluorescence ratio 340/380) in PTH-C1 cells transiently transfected with a scrambled siRNA (control, black, n = 76 cells pooled from 5 independent experiments) or a Trpc1-specific siRNA (siTRPC1, red, n = 61 cells pooled from 6 independent experiments) and cultured in 1, 5, or 10 mM extracellular Ca²⁺. (C) Time course of spermine-induced (5 mM) intracellular Ca²⁺ concentration in PTH-C1 cells transiently transfected with a scrambled siRNA (control, black, n = 144 cells pooled from 4 independent experiments) or a Trpc1-specific siRNA (siTRPC1, red, n = 178 cells pooled from 8 independent experiments). (D) Time course of intracellular Ca²⁺ concentration in PTH-C1 cells cultured in 0 extracellular Ca²⁺ and activated by spermine (3 mM) in the presence (blue, n = 279 cells from 6 experiments) or absence of NPS2143 (300 nM) (black, n = 233 cells from 5 experiments). (E) Time course of R568-induced (50 μM) intracellular Ca²⁺ concentration in PTH-C1 cells transiently cotransfected with GCaMP3 and scrambled siRNA (control, black line, n = 216 cells pooled from 9 independent experiments) or TRPC1 siRNA (siTRPC1, red line, n = 172 cells pooled from 9 independent experiments) in the presence of 1.8 mM extracellular Ca²⁺ concentration. F₀ was the average fluorescence for 1 minute prior to the addition of R-568. (F and G) TRPC1 is specifically coupled to CaSR. HEK293 cells were transiently cotransfected with GCaMP3, CaSR (F), or m1 muscarinic acetylcholine receptor (m1 AchR, G) in the presence or absence of TRPC1 (red or black traces). Cells were first stimulated with thapsigargin (2 μM) to deplete the internal stores and then with spermine (2 mM, F) or carbachol (10 μM, G) to activate CaSR or m1 AchR, respectively. Changes in intracellular free Ca²⁺ concentration are reported as F/F₀. Data were pooled from 6 independent experiments totaling 263 cells transfected with CaSR (black, F) and 316 cells transfected with CaSR plus TRPC1 (red, F) or from 8 independent experiments totaling 190 cells transfected with m1 AchR (black, G) and 173 cells transfected with m1 AchR plus TRPC1 (red, G).