We previously reported that TRPV4 and TRPC1 can co-assemble to form heteromeric TRPV4-C1 channels.In the present study, we characterized some basic electrophysiological properties of heteromeric TRPV4-C1 channels.4α-phorbol 12,13-didecanoate (4α-PDD, a TRPV4 agonist) activated a single channel current in HEK293 cells co-expressing TRPV4 and TRPC1.The activity of the channels was abrogated by a TRPCl-targeting blocking antibody T1E3.Conductance of the channels was ~95 pS for outward currents and ~83 pS for inward currents.The channels with similar conductance were also recorded in cells expressing TRPV4-Cl concatamers, in which assembled channels were expected to be mostly 2V4:2Cl.Fluorescence resonance energy transfer (FRET) experiments confirmed the formation of a protein complex with 2V4:2Cl stoichiometry while suggesting an unlikeliness of 3V4:1Cl or 1V4:3Cl stoichiometry.Monovalent cation permeability profiles were compared between heteromeric TRPV4-Cl and homomeric TRPV4 channels.For heteromeric TRPV4-Cl channels, their permeation profile was found to fit to Eisenman sequence Ⅵ, indicative of a strong field strength cation binding site, whereas for homomeric TRPV4 channels, their permeation profile corresponded to Eisenman sequence Ⅳ for a weak field strength binding site.Compared to homomeric TRPV4 channels, heteromeric TRPV4-Cl channels were slightly more permeable to Ca2+ and had a reduced sensitivity to extracellular Ca2+ inhibition.In summary, we found that, when TRPV4 and TRPCl were co-expressed in HEK293 cells, the predominate assembly type was 2V4:2Cl.The heteromeric TRPV4-Cl channels display distinct electrophysiological properties different from those ofhomomeric TRPV4 channels.