The Pain Pathway. dorsal root ganglion. primary afferent nociceptor. TRP: Transient Receptor Potential

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1 Presented by Issel Anne L. Lim 1 st Year PhD Candidate Biomedical Engineering Johns Hopkins University / Ca Signals in Biological Systems Outline The Pain Pathway TRP: Transient Receptor Potential Experimental Methods and Results TRPM8: Cold Receptor TRPV1: Heat Activation Two-State Model for Voltage-Activated Channel Chemical Agonists 1

2 The Pain Pathway cortex thalamus Exogenous Pain-Producing Stimuli 1) mechanical dorsal root ganglion primary afferent nociceptor projection neuron spinothalamic tract spinal cord 2) chemical < 8 o C > 52 o C 3) thermal 4) electrical Michael Caterina; JHMI; 2006: 1 st Year Medical Student Lecture on Molecular Basis of Pain TRP: Transient Receptor Potential Principal temperature sensors in mammals: transient receptor potential (TRP) superfamily of cation channels Very few positively-charged residues in S4 Possible TRP Mechanisms: Changes in temperature production + binding of channel-activating ligands Channel protein itself temp-dep structural rearrangements that open channel Temp-dependent lipid bilayer rearrangements thermotrps sense changes in membrane tension Intracellular Ca 2+ and membrane depolarization gated openings in cation channels TRPM4 and TRPM5 2

3 TRPM8: Whole-Cell Patch Clamp HEK 293 cells transfected w/ TRPM8 Cooling to 15 o C current Addition of menthol TRPM8: Cell-Free Inside-Out Patches Cold Menthol macroscopic TRPM8 currents Effect of cooling on TRPM8 currents in inside-out patches I/V relations at end of voltage steps 3

4 Rectification: Closing TRPM8 Channel Classical tail current protocol TRPM8 closes at negative voltages Linear I/V relation after prepulse ohmic I/V relation Time-dependent closure at negative potentials w/o Ca 2+ / Mg 2+ in solution divalent cations don t cause rectification TRPM8 = voltage-dependent channel activated by membrane depolarization Outward rectification from rapid and voltagedep closure of channel at negative voltages Voltage Dependence Cold Receptor Does membrane voltage influence cold sensitivity of channel? Measure current activation at different holding potentials during slow cooling of TRPM8- expressing cells Whole-cell TRPM8 currents in response to slow heating of bath solution Normalized current responses as a function of temperature 4

5 Temperature Voltage Dependence Voltage step protocol ( mV) V 1/2 decreased by ~150mV upon cooling and saturated around +25mV btwn 5-10 o C Cooling activates TRPM8 by causing a shift of the voltage dependence of activation TRPV1: Heat Activation Heating activates TRPV1 by shifting the voltage dependence of activation Activation of TRPV1 at depolarized voltages occurred at lower temperatures than at hyperpolarized voltages 5

6 TRPV1: Heat Activation Current traces at diff temps in response to voltage steps Steady-state activation curves at diff temperatures V 1/2 as a function of temperature Temperature Sensing TRPM8 and TRPV1 are activated by temperature changes in cell-free patches Implies that second messengers aren t mechanism for channel inactivation Temp sensitivity modulated by transmembrane voltage Channel activation doesn t result from a temp-dep phase transition of lipid or conformational transition of channel protein 6

7 Temperature Sensing Two-state model for voltage-gated channel E a,open = activation energies associated w/ channel opening α = opening rate β = closing rate R = gas constant (8.31J/(mol K)) T = absolute temperature z = effective charge associated with voltage-dep gating δ = fraction of z moved in outward direction F = Faraday constant (9.65 x 10 4o C/mol) V = transmembrane voltage A, B = preexponential factors TRPM8 & TRPV1: Opening vs. Closing TRPM8: α : shallow temperature dependence E a,open = 15.7kJ/mol w/ Q 10 of 1.2 β : steep temperature dependence E a,close = 173kJ/mol w/ Q 10 of 9.4 TRPV1 α : steep temperature dependence E a,open = 208kJ/mol w/ Q 10 of 14.8 β : shallow temperature dependence E a,close = 23.2kJ/mol w/ Q 10 of

8 TRPM8 and TRPV1: Arrhenius Plots Estimated activation energies not significantly altered by membrane voltage TRPM8 TRPV1 TRPM8: Experimental vs. Model 8

9 TRPV1: Experimental vs. Model Temperature Sensitivity Occurs whenever the activation energies associated w/ the opening and closing transitions are sufficiently different E a,open << E a,close Open probability of channel will increase upon cooling (TRPM8) E a,open >> E a,close Open probability of channel will increase upon heating (TRPV1) 9

10 Ligand Activators TRPM8 and TRPV1 are temperature sensors and ionotropic receptors TRPV1: heating: vanilloids, protons, capsaicin TRPM8: cooling: plant-derived, synthetic, menthol Ligand-Gated Cation Channels Ligand Ligand Na + Ca 2+ Ligand Ligand Ca 2+ depolarization voltage-gated sodium channels vesicle release action potentials transmission to central nervous system neurogenic inflammation pain Michael Caterina; JHMI; 2006: 1 st Year Medical Student Lecture on Molecular Basis of Pain 10

11 TRPM8 + Menthol Menthol significantly shifts cold sensitivity of TRPM8 to higher temperatures Menthol-induced leftward shift of activation curve was independent of temperature TRPV1 + Capsaicin Capsaicin shifts the TRPV1 activation curve 11

12 Conclusions Tight link between temperature sensing and voltage-dependent gating in two thermotrps with opposite temperature sensitivity Thermosensitivity arises from difference in activation energies associated w/ voltagedependent opening and closing Chemical agonists function as gating modifiers to mimic and potentiate thermal responses Membrane voltage contributes to fine-tuning of cold- and heat-sensitivity in sensory cells 12