Figure 2. Functional analysis of the TRPV1^N331K channel.

(A) Western blot analysis of T-REx-293 cells stably expressing hTRPV1^WT (clone 2) or hTRPV1^N331K (clone 2) at 2 different doses and durations of TET incubation, using anti-TRPV1 (α-TRPV1) antibody. α-Actin antibody was used as a protein loading control (n = 3). (B) Traces of Fura-2–based Ca²⁺ imaging from a representative plate of T-REx-293 cell lines stably expressing hTRPV1^WT at LE levels (left trace), hTRPV1^N331K at LE levels (middle trace), and hTRPV1^N331K at HE levels (right trace) in response to application of capsaicin (left and middle, 1 μM; right, 100 μM). Application of the Ca²⁺ ionophore ionomycin (Iono) served as a positive control (middle and right traces). The thick red line represents the mean trace. SES, standard extracellular solution. (C–F) Maximal (max) normalized fluorescence changes from baseline (see Methods) during the application of various TRPV1 agonists. (C) hTRPV1^WT LE, 1 μM capsaicin (Cap) (N = 5 plates, n = 287 cells), hTRPV1^N331K LE, 1 μM capsaicin (N = 3, n = 189), hTRPV1^N331K HE, 100 μM capsaicin (N = 3, n = 178); (D) hTRPV1^WT LE, 10 nM RTX (N = 2, n = 88), hTRPV1^N331K HE, 500 nM RTX (N = 2, n = 100); (E) hTRPV1^WT LE, solution of pH = 4 (low pH, N = 6, n = 340), hTRPV1^N331K LE, low pH (N = 2, n = 161), hTRPV1^N331K HE, low pH (N = 4, n = 232); (F) hTRPV1^WT LE, 2 μM DkTx (N = 2, n = 202), hTRPV1^N331K HE, 5 μM DkTx (N = 2, n = 204), hTRPV1^N331K HE, 5 μM DkTx plus 10 μM capsaicin (N = 2, n = 204). (G) Maximal normalized fluorescence changes from baseline during heat ramp from 25°C to 50°C at various temperatures: hTRPV1^WT LE, heat (N = 4, n = 155); hTRPV1^N331K HE, heat (N = 6, n = 155). ****P < 0.0001, by 2-tailed Student’s t test with Bonferroni’s correction for multiple comparisons (C–F) and 2-way ANOVA with Bonferroni’s correction for multiple comparisons (G).