In neurons, voltage-dependent calcium channels (VDCCs) are involved in the release of neurotransmitters, and in a number other processes in which a rise in intracellular calcium is the primary signal.The function of VDCCs may be modulated by several means including phosphorylation and activation of pertussis toxin-sensitive G proteins by a variety of receptors. Professor Dolphin's group have shown by the microinjection of antisense oligonucleotides in dorsal root ganglion neurons that G protein-alpha(o) subunit is the primary protein involved in coupling GABAB receptors to inhibition of VDCCs, and that the VDCC accessory betha subunit play a pivotal role in the coupling process.The overall results suggest a competition mechanism of activated G protein subunits and VDCC-betha subunits for binding to VDCC-alpha subunits. In the present project we are intended to identify the molecular determinants for such interactions in the subunits of different subtypes of cloned VDCCs and in the G-protein subunits. This will be examined biochemically with the gel overlay technique and using binding assays involving the measurement of protein-protein interaction by binding to scintillant containing beads. Whether the selected sequences disrupt productive G protein coupling to calcium channels will subsequently be studied in an electrophysiological setting, using the patch clamp technique in neurons and neuronal cell lines as heterologous mammaliar expression systems.
Training content (objective, benefit and expected impact)
It is now essential for electrophysiologists working on ion channel biophysics, to have a good grounding not only in the principles of molecular biology and manipulation of DNA, but also in the techniques involved in studying protein-protein interactions. The reason is that one of the main problems facing research on hetero-oligomeric ion channels at the present time are those of specifity of subunit assembly. This project will combine a training in modern molecular biology, biochemistry and cellular electrophysiology (patch clamp) techniques. This will be extremely important to me on my retun a laboratory in Spain. Links with industry / industrial relevance (22)
The proposed supervisor Professor Annette Dolphin has a number of industrial links, with Merck Sharp Dohme Neuroscience Research Center UK and with Glaxo-Wellcome, UK. The industrial relevance concerns the possibility of finding specific neuronal calcium channel blocking drugs, with therapeutic relevance to stroke neurodegeneration and epilepsy.