STUDIES INTO THE MECHANISTIC BASIS OF LOCAL ANESTHETIC ACTION ON TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL SUBFAMILY V MEMBER 1 IN VITRO by RICARDO ENRIQUE RIVERA BSc., The University of Puerto Rico-Cayey, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Pharmacology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2013 © Ricardo Enrique Rivera, 2013 Abstract 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 ii 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. iii Preface The author, Ricardo E Rivera-Acevedo, performed the majority of the research and analysis leading to the results included in this dissertation which is comprised of work that has been published, awaiting publication and currently unpublished. Chapters 2, 4, and 5 represent articles that have been published, and are presented in their original forms, except for the reduction and modification of the introduction and methods sections to preserve continuity. The background for each of these chapters is laid out in the broad introductory section of this thesis. Chapter 3 contains currently unpublished work as of this writing. All the material is presented in the most up-to-date form at the time of completion of this thesis. Additionally, to avoid redundancy, a comprehensive general methods section was included. Xenopus laevis oocytes were used and isolated using methods approved by the UBC Animal Care Committee, certificate #A10-0074. The relative contributions of R.E. Rivera-Acevedo and fellow researchers are summarized below for each chapter: Chapter 2: Rivera-Acevedo, R.E., Pless, S.A., Ahern C.A., Schwarz, S.K.W. The quaternary lidocaine derivative, QX-314, exerts biphasic effects on the transient receptor potential vanilloid subtype 1 channels in vitro. Published: Anesthesiology 114 (2011) pp. 1425-34. Reproduced with permission. R.E. Rivera-Acevedo was responsible for approximately 80% of the work in this Chapter; including designing and performing all experiments, analyzing data, and writing the first draft of the manuscript, as well as re-writing portions of the manuscript requested by journal reviewers. S.A Pless contributed to the overall project design and the manuscript revision. C.A. Ahern was involved in developing the project and revising the manuscript. S.K.W. Schwarz was involved in developing the project and supervising the writing and revising of the manuscript as corresponding author. Chapter 3: Rivera-Acevedo, R.E. Local anesthetics exhibit pH-dependent inhibition of transient receptor potential vanilloid subtype 1 channels in Xenopus oocytes. Chapter 4: Rivera-Acevedo, R.E., Pless, S.A., Schwarz, S.K.W., Ahern C.A. Extracellular quaternary ammonium blockade of transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes. Published: Mol Pharm 82 (2012) pp. 1129-35. R.E. Rivera-Acevedo was responsible for approximately 80% of the work in this Chapter; including designing and performing all experiments, analyzing data, and writing the manuscript, as well as performing additional experiments requested by journal reviewers. S.A Pless contributed to the overall project design and the manuscript revision. S.K.W. Schwarz was involved in developing the project and revising the manuscript. C.A. Ahern was involved in developing the project and supervising the writing and revising of the manuscript as corresponding author. Chapter 5: Rivera-Acevedo, R.E., Pless, S.A., Schwarz, S.K.W., Ahern C.A. Expression- dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes. Channels 7 (2013) pp. 47-50. iv R.E. Rivera-Acevedo was responsible for approximately 60% of the work in this Chapter; including designing and performing all experiments, analyzing data, and writing the manuscript. S.A Pless contributed to the overall project design and the manuscript writing. S.K.W. Schwarz was involved in developing the project and revising the manuscript. C.A. Ahern was involved in developing the project and supervising the writing and revising of the manuscript as corresponding author. v Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ ix List of Figures ................................................................................................................................ x List of Abbreviations ................................................................................................................... xi List of Symbols ........................................................................................................................... xiii Acknowledgements .................................................................................................................... xiv Chapter 1: General introduction ................................................................................................. 1 SCOPE OF TOPIC AND APPROACH TO RESEARCH ........................................................................ 1 OVERVIEW ............................................................................................................................... 4 HISTORY OF LOCAL ANESTHESIA ............................................................................................. 6 Cocaine: the first local anesthetic ..................................................................................... 6 Rise of modern local anesthetics ....................................................................................... 8 LOCAL ANESTHETIC STRUCTURE ACTIVITY RELATIONSHIP (SAR) ......................................... 10 Aryl group ......................................................................................................................... 12 X function ......................................................................................................................... 12 Aminoalkyl group ............................................................................................................. 13 PHYSIOLOGY AND PHARMACOLOGY OF NERVE BLOCK ........................................................... 13 Basic nerve function ........................................................................................................ 13 Molecular mechanisms of local anesthesia .................................................................... 15 Importance of fiber size on function ..............................................................................