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

Abstract

Transient receptor potential vanilloid subtype 1 (TRPV1) channels are essential nociceptive integrators in primary afferent neurons. These nonselective cation channels are inhibited by local anesthetic compounds through an undefined mechanism. Here, we show that lidocaine inhibits TRPV1 channels expressed in Xenopus laevis oocytes, whereas the neutral local anesthetic, benzocaine, does not, suggesting that a titratable amine is required for high-affinity inhibition. Consistent with this possibility, extracellular tetraethylammonium (TEA) and tetramethylammonium application produces potent, voltage-dependent pore block. Alanine substitutions at Phe649 and Glu648, residues in the putative TRPV1 pore region, significantly abrogated the concentration-dependent TEA inhibition. The results suggest that large cations, shown previously to enter cells through activated transient receptor potential channels, can also act as channel blockers.

Permeation and block of TRPV1 channels by the cationic lidocaine derivative QX-314

“…The parent compound of QX-314, lidocaine, also potently inhibits TRPV1 channels (Leffler et al 2008;Rivera-Acevedo et al 2012), but the neutral local anesthetic benzocaine is far weaker, suggesting that it is the charged (protonated) form of lidocaine, similar in structure to permanently charged QX-314, that blocks by binding within the ion permeation pathway (Rivera-Acevedo et al 2012).…”

“…The permeation and block by QX-314 has similarities to TEA ion, which can also carry measurable macroscopic current when present at millimolar concentrations externally (Hellwig et al 2004) and acts as a blocker when applied either internally or externally at submillimolar concentrations (Jara-Oseguera et al 2008;Rivera-Acevedo et al 2012). The entry and exit of TEA and related alkylammonium blockers are influenced by permeating sodium ions in a manner suggesting that the TRPV1 channel is a multi-ion pore (Jara-Oseguera et al 2008;Oseguera et al 2007).…”

Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels inXenopus laevisoocytes

“…[23][24][25] Similarly, density-dependent agonist-sensitivity has been found both in P2X receptors 21 and glycine receptors. 22,26,27 Although we did not observe significant expression-dependent changes in the agonist (capsaicin) sensitivity with TRPV1, 18 we found an inverse correlation between the level of expression and the amount of inhibition by QX-314. Our mutational analysis further pointed toward two amino acids near the putative pore region of TRPV1 (E648 and F649), that may be implicated in the inhibition by QX-314.…”

“…We varied both the amount of mRNA injected, as well as the incubation time (see Methods for details) to obtain oocytes yielding a wide range of maximal current tertiary ammonium compound, lidocaine, and quaternary ammonium compounds such as tetraethyl ammonium (TEA) and tetramethyl ammonium (TMA) can inhibit TRPV1 channels with nanomolar affinity in Xenopus leavis oocytes. 18 In the latter, and in contrast to our initial study, we had limited the whole-cell currents to a range between 0.1 and 3 μA (to limit Ca 2+ overload of the cells due to large inward currents). This led us to speculate that the drastic difference in apparent affinities for ammonium inhibitors may arise from of −60 mV.…”

“…Our mutational analysis further pointed toward two amino acids near the putative pore region of TRPV1 (E648 and F649), that may be implicated in the inhibition by QX-314. 18 Interestingly, the pore region, which generally also determines channel selectivity, has been shown to be a dynamic structure in P2X receptors, [28][29][30] acid-sensing channels 31 and also voltagegated potassium channels, [32][33][34] which have been speculated to bear structural resemblance to TRP channels. In fact, it has recently been shown that TRPV1 can also undergo dynamic changes in the selectivity filter during agonist activation, 35 and importantly, this effect was found to be expression-dependent.…”