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UBC Theses and Dissertations

Studies into the mechanistic basis for local anesthetic action on transient receptor potential cation channel subfamily V member 1 in vitro Rivera, Ricardo Enrique


Transient receptor potential subfamily V, member 1 (TRPV1) channels are important integrators of noxious stimuli with pronounced expression in nociceptive neurons. Local anesthetics have been shown to modulate these nonselective cation channels in vivo and in vitro. However, little is known about the specific interactions between local anesthetic molecules and TRPV1 channels. This thesis therefore was dedicated to examining the mechanistic basis by which local anesthetic compounds act on TRPV1 channels from a perspective of neuronal manipulation for nociceptive blockade. The experimental approach involved a series of in vitro laboratory studies where wild-type TRPV1, TRPV4, and mutant TRPV1 channels were expressed in Xenopus leavis oocytes and cation currents recorded using the two-electrode voltage clamp technique. QX-314 and lidocaine activated TRPV1 channels at millimolar concentrations, but not TRPV4 channels. The TRPV1 antagonist, capsazepine, blocked QX-314- and lidocaine-evoked inward currents through a vanilloid-dependent pathway. At sub-activating concentrations (< 1 mM), QX-314 potently inhibited capsaicin-evoked TRPV1 currents. This thesis’ main results establish that the quaternary lidocaine derivative, QX-314, exerts biphasic effects on TRPV1 channels, inhibiting capsaicin-evoked TRPV1 currents at lower (micromolar) concentrations and activating TRPV1 channels at higher (millimolar) concentrations. Further pharmacological characterization of amino-amide inhibition showed that QX-314 and lidocaine inhibit vanilloid- and proton-evoked currents in TRPV1 channels. Studies defining the molecular determinants of blockade revealed that lidocaine inhibits TRPV1 channels with nanomolar affinity, while the neutral derivative, benzocaine, does not, indicating that a titratable amine mediates the high-affinity block. Consistent with this hypothesis, extracellular tetraethylammonium (TEA) and tetramethylammonium (TMA) application produced potent, voltage-dependent pore block. The overall conclusion is that local anesthetics, previously reported to be able to enter cells through the activated TRPV1 pore, also act as multi-agonist permeant pore blockers. These findings provide an elementary structural model for the molecular interactions between established nerve blocking compounds and the TRPV1 nociceptor. At a fundamental level, this work introduces a novel use for these compounds as molecular probes for the study of TRP channels, whereas at a clinical level, the present results represent a step forward in the development of long-lasting, nociceptive-specific agents for the treatment of pain.

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