(A and B) Gα11 increases TRPC1-mediated Ca²⁺ signaling in HEK293 cells. Cells were transiently transfected with CaSR and TRPC1α or CaSR, TRPC1α, and Gα11. Both groups were cotransfected with the fluorescent Ca²⁺ indicator, GCaMP3, and fluorescence intensity was determined by single-cell Ca²⁺ imaging. Changes in intracellular Ca²⁺ concentration (F/F₀) in response to spermine (1 mM) were determined under physiological conditions (1.8 mM extracellular Ca²⁺, A) or in Ca²⁺-free ECS followed by Ca²⁺ readdition (2 mM, B). Data were pooled from 74 (black) or 85 (red) cells from 2 independent transfections (A) and 318 (black) or 261 (red) cells from 4 independent transfections (B). (C) HEK293T cells were transiently transfected with the long form of TRPC1ε, short form of TRPC1ε, short form of TRPC1α, long form of TRPC1α, or the same TRPC1 plasmids plus myc-tagged Gα11 (top 3 rows) or GαS (bottom 3 rows). Gα11 was immunoprecipitated using α-myc, and GαS was immunoprecipitated using α-GαS. TRPC1 was detected in the complexes using anti-TRPC1 (top). TRPC1 input is shown in middle panels, and immunoprecipitated Gα11 or GαS is shown in bottom panels. (D) Truncation mutants of TRPC1α (GST alone, GST-N(α)-ter, GST-N(ε)-ter, GST-ΔNΔC, GST-C-ter, GST-ΔN) shown in E tagged with GST (blue cylinders) were cotransfected with WT Gα11 or GαS. Protein-protein interactions were determined by GST pulldowns followed by immunoblotting. Gα11 (first panel), but not GαS (third panel), interacted with the cytosolic N- or C-tail of TRPC1. Gα11 input is shown in second panel, GαS input is shown in fourth panel, and immunoprecipitated mutants of TRPC1 are shown.