The molecular mechanisms that mediate mammalian sensory mechanotransduction are poorly understood. Detection of mechanical events by sensory neurons of the dorsal root ganglia (DRG) is the primary event in the senses of touch, pressure-induced pain and proprioception. Recent work has demonstrated that the somatic membrane of cultured DRG neurons is a suitable system for studying physical transduction. In this thesis the responses of cultured DRG neurons to focal mechanical stimulation were investigated. It was shown that mechanical stimulation activated non-selective cation channels in these cells. The response properties of different subclasses of sensory neurons were characterised and were consistent with the presumed in vivo phenotypes of these cells. A number of antagonists of mechanically activated currents, with affnity in the low micromolar range, were identified; these included the pore blocking compounds gadolinium and ruthenium red and FMl-43 acted as a permeant blocker of mechano- sensitive channels. Modulation of mechanically activated currents by extracellular calcium was observed and it was shown that currents were regulated by the actin cytoskeleton and the extracellular matrix protein laminin. Investigation of null mutant mice revealed that the acid sensing ion channels 2 and 3, which are widely hypothesised to function in mammalian mechanosensation, did not contribute to mechanically activated currents. Venom of the marine snail Conus ventricosus was found to block mechanically activated currents although the active component of this venom is yet to be identified. Overall, this work has shown that cultured DRG neurons are a useful system for studying mechanotransduction and has revealed a number of functional and pharmacological properties of the ion channels that underlie this process.

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GPCRs coupled to phospholipase C (PLC) mediate Ins(1,4,5)P3/Ca 2+ release and DAG/PKC activation are capable of generating a multitude of signalling outcomes in vivo. Using a variety of techniques, including single cell imaging, this Thesis will investigate whether Ga q/11PCR specificity is controlled by the activation of particular PLC isoenzymes in a model CHO cell system (CHOm3/alpha1B) and in primary neuronal cells.;The roles of specific PLC isoenzymes were assessed using over-expression or dominant-negative/antisense approaches in the CHOm3/alpha1B cell background. These studies allowed roles for PLCbeta, but not PLCdelta or PLCgamma isoenzymes, to be defined in receptor-mediated responses. Significantly, PLCbeta1 over-expression potentiated Ins(1,4,5)P3/DAG responses to MCh and NA challenge (at sub-maximal agonist concentrations), while PLCbeta3 over-expression attenuated these responses.;The development of rat-specific PLCbeta1 sirRNAs allowed an 80% knockdown of PLCbeta1 protein to be achieved in C6 glioma and cerebellar granule cells. Significantly, M3 mACh receptor-mediated Ca2+ mobilisation was attenuated in C6 glioma cells following siRNA-mediated PLCbeta1 knock-down. In contrast, mACh receptor agonist-stimulated Ins(1,4,5)P3 production was unaffected by PLCbeta1 knock-down in cerebellar granule cells. Further studies revealed that a significant PLCbeta1 knock-down in hippocampal neurones did not attenuate Ins(1,4,5)P3 production mediated by either receptor or concurrent metabotropic/ionotropic stimulation. Interpretation of these data is complicated as PLCbeta3 and PLCbeta4 isoenzymes were shown to be selectively increased by PLCbeta1 siRNA treatment in C6 glioma and cerebellar granule cells, respectively.

This thesis was aimed at the over-expression of the extracellular N-terminal domain (ECD) of the human a7 nicotinic acetylcholine receptor (nAChR). The ECD of the a7 nAChR is more suitable than the ECD of other nAChR subunits for crystallographic studies, mainly because the receptor is oligomeric and does not require any other nAChR subunit ECD to form a complete agonist/antagonist binding domain.