Functional and Modelling Studies of the Transmembrane Region of Trpm8 Channel

Abstract

The Transient Receptor Potential (TRP) ion channel family is ubiquitously present throughout mammalian systems and act as polymodal cellular sensors that measure changes in both global and local environments and respond to a variety of different gating stimuli including intra- and extracellular messengers, chemical, mechanical and osmotic stress, temperature, growth factors and depletion of intracellular Ca2+ stores. Given the importance of Ca2+ signalling in all cell types and the role of TRP channels in regulating Ca2+ homeostasis, it is not surprising that an abnormality in TRP channel function often results in pathogenesis of several diseases including channelopathies like mucolipidosis, polycystic kidney diseases, hypertension and hypomagnesaemia with hypocalcaemia. Within the TRP family, TRPM8 is the cold receptor expressed in DRG neurons, and it is also sensitive to cold mimicking substances such as menthol and icilin. TRPM8 structure has been studied - mainly by point mutagenesis coupled to electrophysiology. TRPM8 monomers associate as homotetramers to form a non-selective cation channel whose selectivity to Ca2+ is about 3 times higher than for Na+, though selectivity filter has not been studied. Little is known on TRPM8 structure-function relation apart that the fourth TM helix (S4) constitutes a moderate voltage-sensing domain and that both cold sensor and PIP2 binding sites are located in the C-terminus of TRPM8. In this study we have used a combination of molecular modelling and experimental techniques to examine the structure of the TRPM8 TM and pore helix regions including conformation of the selectivity filter. We present structural insight into the role of residues involved in intra- and inter-subunit interactions and their link with the channel activity, sensitivity to icilin, menthol, and cold, and impact on channel oligomerization.