The Role of the Selectivity Filter in TRPV1 Channel Gating

Abstract

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 molecules. Although the binding sites for some TRPV1 modulators have been characterized, we still don’t understand how any of these stimuli influence ion conduction in this receptor. The identification and characterization of the regions that function as activation gates are therefore central to understanding the mechanisms of gating in the TRPV1 and other related channels. The structures of TRPV1 in open and closed states suggested that in addition to the intracellular gate formed by the pore-lining S6 helices, the selectivity filter could also function as an activation gate. The available structures of other TRP channels also support the existence of two gates, suggesting that it could be a conserved and unique feature of the TRP family. Here we set out to determine whether the selectivity filter of TRPV1 functions as a gate. We have substituted cysteines along the pore using a cysteineless channel background and assessed their accessibility to externally applied silver ions in both the open and the closed states in patch clamp recordings. First we found that external silver ions act as high-affinity, voltage-dependent, permeant blockers of the cysteineless TRPV1 channel, with a higher affinity for the closed state relative to the open state, suggesting that the filter does not function as a gate. We have provided further support for this hypothesis by showing that accessibility to external silver ions to a cysteine more intracellular than the filter is not affected by channel gating. It is possible that the filter adopts a non-conducting conformation under specific conditions yet to be identified, or that it constitutes a highly dynamic region of the receptor.