Regulation of neurotransmitter release

Dr Talvinder S. Sihra
Lab. Members:
Dr Seraphina Idowu
Ms Jennifer Davies
Ms Tamsin Piper
available for electrophysiologist.

Dr Talvinder S. Sihra is a Senior Lecturer in Pharmacology. He obtained his BSc in Biochemistry and Physiology at the University of Sheffield (1982) and his PhD in Biochemistry at the Department of Biochemistry, University of Dundee (1985). He was Postdoctoral Associate at the Rockefeller University in New York until 1990, when he returned to the University of Dundee with a MRC Fellowship. In 1993, he moved to the Department of Pharmacology, Royal Free Hospital School of Medicine, as a Wellcome Trust Lecturer and in April 1997, he relocated to the Pharmacology Department at UCL, where his laboratory is based in the Medawar Building. He is former secretary of the Neuroscience Group of the Biochemical Society and currently an elected Member of Council. He currently Course Tutor to Joint Physiology and Pharmacology BSc students. He is an Editor for the British Journal of Pharmacology.

1) Presynaptic receptors, through ionotropic and metabotropic mechanisms, represent a fundamental means for regulating neurotransmitter release. One of our interests is to identify and characterize presynaptic receptors that modulate the release of the neurotransmitters glutamate and GABA. The model system we use for these studies is the isolated nerve terminal preparation (synaptosomes). Nerve terminal depolarization leads to Ca²⁺-influx and exocytosis, followed by endocytosis and recycling of transmitter containing small synaptic vesicles (SSVs). To delineate the loci at which presynaptic receptor activation impinge, we use membrane potential-sensitive dyes to assay nerve terminal excitability and depolarization, fura-2 to monitor Ca²⁺-influx and on-line enzymatic assays or HPLC to determine the release of glutamate and GABA by the exocytosis of small synaptic vesicles (SSVs).

Post-translational modification of the proteins involved in the cascade of events leading to neurotransmitter release offers a powerful means of mediating presynaptic plasticity. Thus, one way that presynaptic receptor activation can potentially modulate the properties of proteins involved in neurotransmitter release is through the stimulation of second messenger cascades that lead to protein phosphorylation or dephosphorylation. Using synaptosomes labelled with ³²P-orthophosphate, we can ascertain presynaptic receptor-mediated activation of specific protein and lipid kinases and phosphatases employing identified intraterminal substrates for these enzymes. Currently, we are characterising the nerve terminal modulatory roles of mitogen-activated protein kinases and lipid kinases leading to the production of polyphosphoinositides.

2) The second major focus of the laboratory is to determine the role of specific protein kinases or phosphatases in the cascade of events leading to SSV exocytosis and endocytosis. For these studies, we have taken the approach of altering enzyme expression in neuronal cell lines and primary cell cultures that are amenable to molecular biological procedures. Currently, we are evaluating the effects of altered expression of the major Ca²⁺-dependent protein phosphatase, protein phosphatase 2B (calcineurin, CN) in the neuroblastoma x glioma cell-line NG108-15. Thus we have transfected NG108-15 cells with expression vectors containing sequences of CN (CN-A subunit) cDNA in sense and anti-sense orientations, to respectively overexpress or denude CN. Stable, individual clones now allow the specific characterization of the role of the enzyme in: (a) controlling voltage-dependent Ca²⁺-influx and, (b) the exocytotic/endocytotic cycling of SSVs and control thereof by the CN substrates, synapsin I and dynamin. We are examining the effect of CN overexpression or diminution on VDCC activity using whole-cell patch-clamping of wild-type and mutant NG108-15 cells. In parallel experiments, the effects of altered CN-expression on Ca²⁺-influx are being subcellularly localised by Ca²⁺-imaging of single cells using fura-2. Finally, the action of CN at the level SSV-associated proteins is being assessed using the SSV probe FM1-43 to image endocytotic/exocytotic events at the single cell level.