Expression and function of TRP channels in peripheral sensory neurons

Sammanfattning: Transient receptor potential (TRP) channels comprise a family of multifunctional proteins which has since its discovery been of particular interest in sensory biology even though they are also frequently expressed in non-neuronal cell types. It is evident that TRP channels play an important role in the function of sensory neurons, not at least by being sensors of external stimuli. Direct activation by thermal and chemical stimuli has been shown while their involvement in mechanosensation is still elusive. In addition many TRP channels also seem to have more modulatory roles in the cell by for example adjusting intracellular Ca2+ levels in response to activation of other membrane proteins. This thesis presents new findings on the expression and function of TRP channels in peripheral sensory neurons. Using quantitative real-time PCR the developmental expressions of all 28 vertebrate TRP channels were studied. Results showed that most channels are expressed in adult thoracic and lumbar dorsal root ganglia (DRG) and in nodose ganglia (NG). The most common expression pattern displayed low levels at early embryonic development followed by progressively increasing levels at later stages of development, reaching its highest expression at postnatal and adult stages. Cellular localisation studies of selected channels in the DRG further revealed expression in different neuronal subtypes. TRPC1 and TRPC2 were primarily found in large sized mechanosensitive neurons, TRPC3 was exclusively found in non-peptidergic nociceptors and TRPM8 was found in a very limited population of small sized neurons, not co-labelling with any of the tested markers for neuronal subtypes. These findings open for potential new, previously undefined roles, for some of the TRP channels in sensory neurons. To elucidate new functional roles, an expression study measuring differential regulation of all TRP channels in rats during development of neuropathic pain was conducted. Downregulation was evident for TRPM6, A1, V1, M8, C3, C4 and C5 while upregulation was seen for TRPML3. TRPM6, C3, C4, C5 and ML3 have never previously been associated with pain and are therefore considered to be important new findings. Since the identification of TRPC1 as a stretch sensor in liposomes from oocytes this finding has been questioned due to difficulties in confirmation of these results in heterologous cell systems. To further explore this issue we used native neurons from DRG to study the possible involvement of TRPC1 in mechanosensitivity. In neurons subjected to short hairpin RNA (shRNA) mediated knockdown of TRPC1, the response to cell stretch was shown to be reduced by 65%. These data further emphasise the indication that TRPC1 is important, either directly or indirectly, in the activation mechanism of mechanosensation. In conclusion, this thesis show that most of the 28 vertebrate TRP channels are expressed in peripheral sensory ganglia and have specific neuronal subtype expression patterns. Furthermore, the data suggest potential new roles related to pain pathophysiology for several TRP channels and further strengthen the hypothesis of TRPC1 involvement in mechanosensation.