TRPV1 is a non-selective cation channel thought to play a key role in the detection of noxious stimuli by sensory neurons. TRPV1 effectively serves as a molecular integrator of a number of diverse activators including capsaicin, protons and heat (>43°C), however, the physiological (and pathophysiological) activators of TRPV1 in vivo are still debated. Protons are known to both directly activate TRPV1 and to cause potentiation of heat or capsaicin-mediated agonism. Consequently, they are likely to be a key endogenous regulator of TRPV1. We have therefore undertaken a detailed investigation of the effects of protons on the hTRPV1 expressed in HEK293 cells using the whole-cell patch clamp technique. Application of protons was achieved by applying a solution of known pH (9.3-4.3) to the cell under study using a rapid perfusion system (see Hayes et al. 2000). Data are given as mean ± S.E.M. and significance (P < 0.05) was ascertained using Student’s unpaired t test.
Protons behaved as an agonist on hTRPV1 expressing cells leading to the activation of slowly desensitising currents (pEC50 = 5.21) which were inhibited by the TRPV1 antagonists capsazepine and SB-366791 (1 µM). Acid-evoked currents deactivated rapidly upon agonist removal commensurate with a highly cooperative gating mechanism and an externally accessible binding site. Interestingly, a degree of apparent proton-mediated inhibition was also noted at pH ▓le│ 5. Acidification also caused marked potentiation (from 3.27 ± 0.14, n = 8, to 13.44 ± 6.20 fold, n = 7, at pH 6.5 and pH 4.3, respectively) of capsaicin-gated currents. This potentiating effect was estimated to be half maximal at ~pH 6.0. Equivalent potentiation by pH 6.5 was observed at supramaximal concentrations of capsaicin (3.08 ± 0.03 fold at 30 µM) and other agonists such as olvanil (100 nM, 3.54 ± 0.11 fold, n = 5) and resiniferatoxin (100 nM, 3.16 ± 0.08 fold, n = 5). These data are consistent with a mechanism in which protons mediate a distinct agonist-independent effect on TRPV1 function rather than by altering agonist potency. In further experiments, we noted that the degree of potentiation by protons was related to the rectification properties of hTRPV1 and demonstrated that the acid-mediated potentiation of capsaicin-gated currents was itself strongly voltage-dependent.
In summary, we have shown that proton modulation of the hTRPV1 receptor is complex, with extracellular acidification causing response potentiation, direct gating and a degree of current block as pH is progressively lowered. Our findings imply that the voltage-and time-dependent properties of the receptor and the potentiation of hTRPV1 by protons may be inextricably linked by a common mechanism.