Unraveling Allosteric Coupling Mechanisms in the TRPV1 Channel

The TRPV1 channel is a homotetrameric non-selective cation channel that functions in nociceptors as an integrator of external noxious stimuli and endogenous pro-inflammatory signaling molecules. The diversity of its modulators is staggering, including voltage, lipids, protons, cations, temperature, protein kinases, oxidizing and reducing agents and protein toxins. Structural perturbations throughout the receptor and some antagonists have been shown to selectively ablate specific modalities of channel activation without entirely disrupting others, suggesting that most stimuli use distinct molecular mechanisms for regulating the channel. Nevertheless, most stimuli that activate TRPV1 are strongly coupled, exhibiting synergy in both their efficacy and affinity. Here we studied the effects of small extracellular monovalent cations, which had been shown to inhibit channel activity, on temperature-, proton- and capsaicin-dependent activation of the TRPV1 channel. We found that substitution of extracellular sodium or potassium with N-methyl-D-glucamine has a strong effect on channel temperature sensitivity, shifting the threshold of activation to lower temperatures by > 20°C and causing a 5-10-fold reduction in Q10. However, we also found that both heat and capsaicin can overcome the inhibitory effects of sodium and lead to channel activation in a sodium-independent manner once the cation-inhibitory site has reached saturation, suggesting these stimuli act through distinct mechanisms. In contrast, the potentiating effect of protons at pH < 6 seems to be dependent on sodium concentration. We are currently performing experiments under elevated sodium conditions on proton-associated TRPV1 mutants to determine whether protons modulate the channel by two different mechanisms that cause channel activation or potentiation, and whether sodium is involved in either of these.