Modulation of neuronal excitability by heterotrimeric G-proteins and Rho GTPases

Nerve cells communicate through electrical signals termed action potentials. These are generated by ion channels, which are specialized protein molecules in cell membranes. Individual ion channels are on/off switches that control the flow of ions across the membrane. When switched on, they allow a specific type of ion / Na+, K+, or Cl- - to cross the membrane, thereby producing small electrical currents that underlie action potentials and transmit information from one place to another. Action potentials in neurons are very brief and constitute the currency of rapid neuronal signaling. However, some ion channels in nerve cell membranes can generate smaller, but longer lasting changes in the membrane potential. Their function is to regulate the frequency and pattern of action potential discharges on a slower time-scale. Some of these channels can respond to hormones and neurotransmitters released by neighbouring nerve cells, and thereby change the sensitivity of neurons to incoming signals and their responses. This phenomenon is known as 'neuromodulation'. The fine tuning of neuronal excitability by neuromodulatory transmitters represents the cellular correlate of higher mental functions such as arousal or paying attention. This programme of research focuses on the neuromodulatory effects of two important chemical transmitters in the brain: acetylcholine and glutamate. Our aim is to understand the signalling events taking place in single neurons and leading from the binding of these transmitters to specific receptor proteins (muscarinic and metabotropic glutamate receptors) to changes in the activity of ion channels. In particular, we are interested in identifying and studying the function of 'large' and 'small' G-proteins. G proteins are perhaps the most important signal transducing molecules in cells, translating signals into biological effects inside cells. They are fundamental components of the pathways linking neurotransmitter receptors to ion channels. Our previous work and preliminary data indicate that acetylcholine and glutamate activate different types of 'large' and 'small' G-proteins in neurons. Electrophysiological techniques in single neurones from normal and genetically modified mouse brains, together with molecular biological and biochemical techniques, will be used to study the function of these 'large' and 'small' G-proteins in the pathways linking the neurotransmitter receptors to ion channels and, ultimately, neuronal firing behaviour. The outcome of our research should further our understanding of the molecular and cellular mechanisms underlying the extraordinary range of cognitive and behavioural, functions exerted by acetylcholine and glutamate through muscarinic and metabotropic glutamate receptors in the brain. Additionally, in the long term, it might provide novel potential drug targets, given that alterations in the signalling by these receptors have been implicated in the pathophysiology of several major diseases of the central nervous system, including Alzheimer's and Parkinson's disease and anxiety disorders.

Acetylcholine and glutamate, through metabotropic receptors, play a crucial role in generating network oscillations, plasticity and in learning and memory in the hippocampus. An important target for their neuromodulatory actions is the slow calcium-activated potassium current (sIAHP) that shapes the firing pattern of cortical neurons. Acetylcholine and glutamate suppress this current, thereby enhancing neuronal excitability. However, the signalling mechanisms mediating this effect are still largely unknown. This proposal aims to identify the G proteins that mediate the cholinergic and glutamatergic modulation of sIAHP. We have previously demonstrated a role for Gq, and preliminary results suggest the involvement of a small G-protein of the Rho family. Our first aim is to study the function of RhoA and other small G-proteins in the cholinergic and glutamatergic inhibition of sIAHP in hippocampal neurons. We will use a combination of electrophysiological and biochemical approaches to determine whether Gq and RhoA act in a common signalling pathway. If evidence is obtained for a direct Gq/RhoA interaction, this will be further characterized by fluorescence methods in live cells. Alternatively, additional components of the Gq/RhoA complex will be identified biochemically. We have shown that Gq mediates only part of sIAHP inhibition by acetylcholine and glutamate, suggesting the existence of a parallel signalling pathway. Our second aim is to analyse the role of G12/13 proteins in this hypothetical pathway by analysing mouse mutants lacking G12/13 globally, or specifically in the brain. Biochemical experiments will further elucidate whether G12 or 13 are functionally coupled to RhoA. This study may reveal novel functions for Rho GTPases and G12/13 in neuronal signalling. This will increase our understanding of the molecular mechanisms used by acetylcholine and glutamate to activate distinct signalling pathways and regulate complex behavioural and cognitive processes.