Characterization of phospho-disruptive TRPV1 mutant

The rat TRPV1 cDNA (in pcDNA3) was generously provided by Prof. David Julius. Site-directed mutagenesis was performed on rTRPV1-pcDNA3 to generate the phospho-disruptive alanine substitution at PKC phosphorylation sites, S502, T704 and S800 (rTRPV1-S502A/T704A/S800A or rTRPV1-TM as denoted in the figure), as described previously (Mohapatra et al., 2003 (link); Loo et al., 2012 (link)). Mutations at only these three residues were confirmed by DNA sequencing of the full cDNA. Human embryonic kidney cells stably expressing the T-antigen (HEK293T) were cultured in DMEM (with Glutamax), 10% FBS and penicillin/streptomycin. Cells were co-transfected with plasmids containing eYFP-N1 (Clontech) and wild-type (WT) or PKC-triple mutant TRPV1, and/or YFP-tagged rat PTH1 (generously provided by Prof. Matthew Mahon) cDNAs using the Lipofectamine-2000™ reagent per the manufacturer’s instructions, as detailed previously (Mohapatra and Nau, 2003 (link), 2005 (link); Loo et al., 2012 (link)). Transfected cells were used for recordings 1–2 days post-transfection.