FIGURE 3.

(−)-EA activates heteromeric channels containing TRPC4 and TRPC1. A, schematic depicting the design of a TRPC4-TRPC1 concatamer with N-terminal TRPC4β (893 amino acids) linked to C-terminal TRPC1 (759 amino acids) via a flexible eight-amino acid linker. B, Western blot with anti-TRPC1 antibody confirming tetracycline (Tet)-inducible expression of the full-length concatamer protein (predicted size, 191 kDa). Control HEK T-REx cells expressing monomeric TRPC1 protein are provided for comparison. IB, immunoblot. C, (−)-EA current in HEK T-REx cells expressing the TRPC4-TRPC1 concatamer. A whole-cell voltage clamp recording of membrane current during ramp changes in membrane voltage from −100 to +100 mV applied every 10 s is shown. Only currents sampled at −100 and +100 mV are displayed. 100 nm (−)-EA was bath-applied as indicated by the horizontal bar. D, full current traces during two ramp changes in voltage, one during the initial application of vehicle (DMSO) and the other after application of (−)-EA. E, example of 96-well plate fura-2 measurements of the change (Δ) in the intracellular Ca²⁺ concentration evoked by (−)-EA in TRPC4- and TRPC4-TRPC1 concatamer (TRPC4/C1)-expressing HEK T-REx cells (n = 4). F, mean data for experiments of the type shown in E measured between 1 and 2 min after application of a range of (−)-EA concentrations. Error bars indicate S.D., and the fitted curves are Hill equations (N = 3).