Search

TRPM8 is the principal sensor of cold temperatures in mammalian primary sensory neurons. Cold temperatures 28~8¿¿¿¿C and the cooling compound menthol activate TRPM8. TRPM8 is expressed on nociceptive and non-nociceptive primary sensory neurons and mediates innocuous and painful cold sensations. Using calcium imaging, I examined menthol responses and role of protein kinases in two functionally distinct populations of cold-sensing DRGs that use TRPM8 receptors to convey innocuous (menthol-sensitive/capsaicin-insensitive, MS/CI) and noxious (menthol-sensitive/capsaicin-sensitive, MS/CS) cold sensation. PKC activation decreased menthol response in all neurons. MS/CI neurons had larger menthol responses with greater adaptation and adaptation was attenuated by blocking PKC and CaMKII. In contrast MS/CS neurons had smaller menthol responses with less adaptation that was not affected by blocking PKC or CaMKII. In both MS/CI and MS/CS neurons, menthol responses were not affected by PKA activation or inhibition. Taken together, these results suggest that TRPM8-mediated responses are different between non-nociceptive-like and nociceptive-like neurons (Chapter II). Calcium influx causes a feedback regulation of TRPM8 currents that when analyzed under whole-cell voltage-clamp exhibit a Ca2+-dependent functional downregulation with two distinctive phases, a shorter, faster acute desensitization and a prolonged tachyphylaxis. Using acutely dissociated rat DRGs I examined TRPM8 whole-cell currents while pharmacologically manipulating several intracellular targets. TRPM8 acute desensitization is caused by calmodulin and requires phosphatidylinositol 4,5-bisphosphate (PIP2). Conversely, tachyphylaxis is mediated by hydrolysis of PIP2 and activation of PKC/phosphatase 1,2A. Consequently, I set out to determine the mechanisms underlying the mentioned findings by studying inside-out recordings of TRPM8 channels stably expressed in HEK 293 cells. PIP2 switches TRPM8 channel gating to a high open probability state with short closed times and Ca2+-calmodulin reverses the effect of PIP2, switching channel gating to a low open probability state with long closed times. Thus, through gating modulation, Ca2+-calmodulin provides a mechanism to rapidly regulate TRPM8 functions in the somatosensory system (Chapter III).It is not well understood how cooling temperatures have multiple sensory effects ranging from generating cooling or painful cold sensation to modifying sensory modalities like touch, itch and pain. With electrophysiology I studied how temperature modulates excitability in DRGs. Cooling temperatures differentially modify the excitability of non-nocicepetive and nociceptive neurons. Cold aborts repeated action potential firing in non-nociceptive neurons by increasing the voltage-dependent inactivation of TTXs Na+ channels and reducing A-type K+ currents. Cooling temperatures also inhibit IA in nociceptive-like neurons, which possessed TTXr Nav 1.8 channels, but these neurons largely retain or increase firing rate. Cold had less inhibition on TTXr Na+ channels, allowing nociceptive neurons to fire at painful cold temperatures. Like cold, IA blocker 4-AP reduced IA-K+ currents in TTXs and TTXr cells, but this led to higher spike frequency only in the latter. Finally, the molecular determinants for neuron excitability under cooling temperatures play a role in defining temperature threshold and ranges for which innocuous and noxious cold directly elicit impulses in nociceptive and non-nociceptive cold-sensing neurons respectively, providing a molecular mechanism for sensory distinction between innocuous and noxious cold stimuli (Chapter IV).