Characterisation of the transduction mechanisms and molecular correlate of the neuronal chloride current, I_Cl(m)

Cl- ions are the most abundant anion species in cells, Cl- channels are important for a range of cellular processes, from the regulation of cell volume and intracellular pH to the modulation of electrical excitability. Sympathetic neurons of the rat superior cervical ganglion (SCG) display a Cl- conductance that is activated by acetylcholine. Concomitant activation of nicotinic and muscarinic receptors is required for full activation, with protein kinase C (PKC) being central to this process. This Cl- conductance is known as ICl(m) and is the main focus of the present study. ICl(m) has a delayed onset (5-10 secs) following a rise in intracellular calcium concentration ([Ca2+]) at physiological temperatures, thus the activation mechanism of this conductance differs from other Ca2+-dependent Cl- currents previously described. The first part of this thesis describes the characterisation of the transduction mechanism involved in ICl(m) activation. Membrane currents were recorded in voltageclamp and intracellular [Ca2+] was monitored using Fura-2. The actin cytoskeleton and A-kinase anchoring protein (AKAP150) were shown to be involved in the local signalling required for ICl(m) activation. ICl(m) was found to be present in sensory neurons as well as sympathetic neurons. Recordings of ICl(m) from intact ganglia suggesting that the current is present physiologically. The current was also observed in a sub-population of dorsal root ganglion (DRG) neurons, where its occurrence was significantly increased after several days in vitro. This indicates that ICl(m) could play a part in pathological or developmental states. The final part of this study aimed to identify the molecular correlate for ICl(m). Over-expression of the candidates was achieved by intranuclear injection of cDNA plasmids, whilst ‘knockdown’ was investigated using siRNA. The strongest candidate emerged as the Ca2+-dependent Cl- channel, TMEM16A, although further studies are required to make an unambiguous conclusion