Properties of the Inner Pore Region of TRPV1 Channels Revealed by Block with Quaternary Ammoniums

Abstract

The transient receptor potential vanilloid 1 (TRPV1) nonselective cationic channel is a polymodal receptor that activates in response to a wide variety of stimuli. To date, little structural information about this channel is available. Here, we used quaternary ammonium ions (QAs) of different sizes in an effort to gain some insight into the nature and dimensions of the pore of TRPV1. We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers. We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics. TEA does not interfere with the activation gate, indicating that this molecule can reside in its blocking site even when the channel is closed. The dependence of the rate constants on the size of the blocker suggests a size of around 10 Å for the inner pore of TRPV1 channels.

Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1

“…This picture showed that the TRPV1 channel shares many structural features with other ion channels of known structures, such as the voltage-gated Kv1.2 channel and the Kv1.2-2.1 paddle chimera (Long et al, 2005a(Long et al, , 2007. The TRPV1 channel structure supported previous studies proposing that the TRPV1 channel is assembled as a tetramer (Kedei et al, 2001;Kuzhikandathil et al, 2001) and confirmed functional studies showing that the channel pore and corresponding intracellular activation gate(s), formed by the S5 to S6 transmembrane segments, exist along the tetramer axis of symmetry (Oseguera et al, 2007;Jara-Oseguera et al, 2008;Salazar et al, 2009). The TRPV1 structure shows that a peripheral domain in each subunit, encompassing S1-S4 segments, is connected to the pore by a short linker, a feature also conserved in voltagedependent potassium channels (Long et al, 2005a).…”

Extracellular Quaternary Ammonium Blockade of Transient Receptor Potential Vanilloid Subtype 1 Channels Expressed in Xenopus laevis Oocytes

“…Taken together, the data available to date indicate that QAs may act as permeant blockers of TRP channels. Consistent with this possibility, TEA and TMA displayed strong voltage-dependent relief of inhibition at Ϫ25 mV compared with that at Ϫ60 mV, similar to the extracellular open channel block of voltage-gated potassium channels and intracellular pore block in TRPV1 channels by other QAs (Jara- Oseguera et al, 2008).…”

“…We thus believe that although the specific drug-receptor affinity may change depending on the expression system (Lev et al, 2012), the observation of QA blockade will be universal, regardless of the cell system used. This notion is further supported by the observation that LAs and QAs can block TRPV1 in mammalian cells (Oseguera et al, 2007;Jara-Oseguera et al, 2008;Leffler et al, 2008).…”

“…In Xenopus laevis oocytes, we have found that lidocaine and QX-314 exert biphasic effects on TRPV1 channels, inhibiting capsaicin-evoked TRPV1 currents at lower (micromolar) concentrations and activating TRPV1 channels at higher (millimolar) concentrations. Whereas the mechanisms are unknown, studies into the permeation and gating pathway of TRPV1 have found, similar to results obtained with Kv channels, that permanently charged quaternary ammonium compounds are able to potently block TRPV1 intracellularly, thus delimiting similarities among these seemingly disparate family members (Oseguera et al, 2007;Jara-Oseguera et al, 2008). However, it remains unclear whether TRPV1 is also susceptible to extracellular inhibition by quaternary ammonium compounds.…”

“…To more directly assess the role of charge in TRPV1 inhibition, we characterized extracellular block by the quaternary permanently charged amines, TEA and TMA, which have been instrumental in the structure-function study of potassium channel pore regions. More recently, these compounds have been successfully used to help define the structure and basic biophysical properties of the intracellular TRPV1 channel pore (Oseguera et al, 2007;Jara-Oseguera et al, 2008). In our current study, similar to results with both lidocaine and QX-314, extracellular application of TEA produced a reduction in capsaicin-evoked inward currents, albeit with substantially higher affinity (Fig.…”