TRPV1 Is Activated by Both Acidic and Basic pH

TRPV1 is activated by intracellular alkalization. A, NH₄Cl (50 mm) increases channel activity recorded in a cell-attached patch from TRPV1-expressing HEK cells (fold change, 4.1 ± 0.8, n = 5) in a BCTC-sensitive manner. Membrane patches were challenged with a voltage ramp protocol (B; 0.25 Hz), and cells were maintained in high extracellular K⁺ (see supplemental methods). The response onset to NH₄Cl was rapid (20 ± 3 s; n = 5). Thirty consecutive sweeps were averaged before (orange) and after (red) activation by NH₄Cl. BCTC (300 nm) inhibited the activation of channel activity by NH₄Cl when cells were preincubated in BCTC (blue trace is a typical example). Vector transfected cells revealed little/no effect of 50 mm NH₄Cl (black). B, pH and BCTC dependence of TRPV1 activity in excised inside-out membrane patch. TRPV1 activity is observed at positive potentials in control conditions (pH 7.3, 25°C; black). Subsequent bath application of pH 7.8 (light blue traces), pH 8.3 (blue) and pH 9.5 (dark blue) increased channel activity at +120 mV by 5-, 40- and 45-fold, respectively. The latency to TRPV1 activation by pH 7.8 and pH 8.3 was 50 ± 13 s (n = 5) and 18 ± 2 s (n = 8). The TRPV1 antagonist BCTC (300 nm) nearly abolished the activity in the pH 9.5 solution (pink), and block was partially reversible upon washout of the antagonist at pH 9.5 (dark blue). The effect of base on TRPV1 was reversed completely upon return to control pH 7.3 solution (black). Representative traces are shown (every 16–20 s) during the peak of each effect. C, D, Whole-cell conductance was monitored from a cell expressing rat TRPV1 at 0.2 Hz over ∼50 min after achieving the whole-cell configuration with a pipette containing pH 8.3 (pipette resistance, 1.7 MOhm; voltage protocol in 2D). Voltage ramp-induced currents were acquired every 5 s from a holding potential of −50 mV. The membrane potential was stepped to −120 mV, ramped to +120 mV at a speed of 2.85 mV/ms and maintained at +120 mV for 50 ms. Outwardly rectifying currents (control a shown in green in D) were measured at +120 mV and −120 mV and the time course plotted (C). After a couple of minutes (data not shown, see below), outwardly rectifying currents increased (b indicated in C, blue trace in D) and were reversibly blocked by bath application of 150 nm BCTC (c indicated in C, black trace in D). After washout (d in C, gray trace in D), capsaicin (CAPS, 1 mm) was bath applied and elicited large outwardly rectifying currents with substantial inward current (e in C, red trace in D) which reversed upon washout. Upon patch rupture, a transient increase in conductance was observed (data not shown) that was dependent on rTRPV1 expression and was independent of the pH used to backfill the pipette since it was also observed in cells with pipettes filled with neutralized IS or standard IS.

Activation of TRPV1 by NH₄Cl or intracellular basic pH is dependent on a single histidine residue H378. A, The mutant TRPV1-H378Q does not respond to NH₄Cl. FLIPR dose responses to NH₄Cl, normalized to peak capsaicin response, are shown for WT TRPV1 and TRPV1-H378Q. B, TRPV1-H378Q shows minor differences in the capsaicin dose-response. FLIPR dose responses, normalized to peak capsaicin response, are shown for TRPV1 (EC₅₀ 12 nm ± 0.7 nm) and TRPV1-H378Q (EC₅₀ 16 ± 1 nm). C, Whole-cell patch-clamp experiments show that TRPV1 and TRPV1-H378Q have identical capsaicin concentration dependence. Whole-cell current density elicited by 5, 10, 30 and 300 nm capsaicin are shown for TRPV1 (blue; EC₅₀ 41 ± 10 nm) and TRPV1-H378Q (red; EC₅₀ 33 ± 16 nm) (3–7 data points per concentration). D, TRPV1-H378Q responds normally to heat and acid. Peak responses to heat (heat ramp, 25–48°C) or acid (pH 4.5–5) normalized to peak capsaicin (1 μm) response for HEK cells expressing TRPV1 or TRPV1-H378Q, using ratiometric calcium imaging. E, TRPV1-H378Q is severely impaired in its response to alkaline pH. Basic pH was applied to the cytoplasmic side of excised inside-out membranes and average channel activity was measured at +120 mV in the absence and presence of basic solutions at the indicated pH. The fold increases in activity for TRPV1 and TRPV1-H378Q are significantly different for all basic solutions. *p < 0.05, **p < 0.005, ***p < 0.001. a.u., Arbitrary unit.