4 YEAR PhD IN NEUROSCIENCE
Department of Neuroscience, Physiology & Pharmacology
Voltage-dependent calcium channels
Voltage-dependent calcium channels are essential for synaptic transmission, providing the Ca2+ for transmitter release. Their modulation provides the basis for an important form of presynaptic inhibition. Furthermore, there are also numerous mutations in Ca2+ channel subunits in humans and mice that result in neurological syndromes including epilepsy and cerebellar ataxia. We study several aspects of voltage-dependent calcium channels, their expression, composition, biophysical properties and modulation by G proteins. We wish to understand how the channels are differentially targeted and fulfill their diverse roles in neuronal function.
The α1 subunits are the pore forming subunits of voltage-gated calcium channels. β subunits are cytoplasmic subunits, and have profound effects on calcium channel expression and properties, and on G protein-mediated calcium channel inhibition. The α2δ subunits are largely extracellular and have their main effect on calcium channel trafficking, with more subtle effects on calcium channel properties.
We are particularly interested in α2δ subunit interactions and how they affect function, since certain α2δ subunits are the targets of the gabapentinoid drugs (Field et al, 2006) and α2δ-1 is strongly upregulated in neuropathic pain (Bauer et al., 2009). The gabapentinoid drugs appear to have a novel mechanism of action, reducing the trafficking of α2δ subunits (Hendrich et al 2008).
Three month project placement for Wellcome Neuroscience PhD students.
Example Project 1: Investigation of cell surface expression of calcium channel subunits in neurons.
Investigation of cell surface expression of calcium channel subunits in neurons.
We will replace an external HA motif with another motif in an external epitope of an α1 subunit and an α2δ subunit that will enable the proteins to be labeled directly with a fluorophore. We will test the construct by expression in Cos7 cells, and then use expression in neurons for further studies
The methods used will involve molecular biology and confocal imaging. You will also learn to culture neurons for expression studies by microinjection or nucleofection.
If the project was extended to a PhD you would perform live cell imaging studies in neurons and examine the effect of gabapentin on the trafficking of the α2δ subunit.
Example project 2: Mutation of the calcium channel subunit α2δ-3, to produce a gabapentin-sensitive subunit.
Gabapentin and its analogues are used extensively for the alleviation of neuropathic pain and also as anti-epileptic drugs. They bind to α2δ-1 and -2, but not to α2δ-3, but their exact mechanism of action is unknown. We predict that a point mutation in α2δ-3 will make it gabapentin-sensitive.
You will make a point mutation in α2δ-3 and examine its gabapentin sensitivity. You will examine the expression of the wild type and gabapentin-sensitive mutant.
Methods; The methods used will involve molecular biology, ligand binding and confocal imaging. You will also learn to culture and transfect cells for expression studies.
Bauer CS, Nieto-Rostro M, Rahmin W, Tran-Van-Minh A, Ferron L, Douglas L, Kadurin, I, Sri Ranjan Y, Fernandez-Alacid L, Millar NS, Dickenson AH, Lujan R and Dolphin AC (2009)..
Journal of Neuroscience 29:4076–4088
Hendrich J, Tran Van Minh A, Heblich F, Nieto-Rostro M, Watschinger K, Striessnig J, Wratten J, Davies A and Dolphin AC (2008)
Pharmacological disruption of calcium channel trafficking by the α2δ ligand gabapentin.
Proc Natl Acad Sci U S A. 105: 3628–3633.
Davies A, Douglas L, Hendrich, J, Wratten J, Tran-Van-Minh, A, Nieto-Rostro M, Pratt, WS, Saibil, HR and Dolphin AC (2006)
The calcium channel α2δ-2 subunit partitions with CaV2.1 into lipid rafts: implications for function and localization.
Journal of Neuroscience 26:8748-8757
Donato R, Page KM, Koch D, Nieto-Rostro M, Foucault I, Davies A, Wilkinson T, Rees M, Edwards FA and Dolphin AC. (2006)
The ducky2J mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression.
Journal of Neuroscience 26: 12576-86.
Field,M.J., Cox,P.J., Stott,E., Melrose,H., Offord,J., Su,T.Z., Bramwell,S., Corradini,L., England,S., Winks,J., Kinloch,R.A., Hendrich,J., Dolphin,A.C., Webb,T., and Williams,D (2006)
Identification of the α2δ-1 subunit of voltage-dependent calcium channels as a novel molecular target for pain mediating the analgesic actions of pregabalin.
Proc. Natl. Acad. Sci. (USA) 103, 17537-17542
Canti C, Nieto-Rostro M, Foucault I, Heblich F, Wratten J, Richards MW, Hendrich J, Douglas L, Page KM, Davies A, Dolphin AC (2005)
The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α2δ subunits is key to trafficking voltage-gated Ca2+ channels.
Proc Natl Acad Sci U S A. 102: 11230-11235
Leroy J, Butcher AJ, Richards M, Davies A, Nieto-Rostro M, Pratt WS and Dolphin AC (2005)
Interaction via a key tryptophan in the I-II linker of N-type calcium channels is required for beta1 but not for palmitoylated beta2, implicating an additional binding site in the regulation of channel voltage-dependent properties.
Journal of Neuroscience: 25: 6984-96
Viard P, Butcher A, Halet G, Davies A, Nürnberg B, Heblich F and Dolphin AC (2004)
PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane.
Nature Neuroscience 7: 939-946
Moss FJ, Viard P, Davies A, Bertaso F, Page KM, Graham A, Cantí C, Plumpton M, Plumpton C, Clare JJ and Dolphin AC (2002)
The novel product of a five-exon stargazin-related gene abolishes CaV2.2 calcium channel expression.
The EMBO Journal 21: 1514-1523
Barclay, J. Balaguero, N., Mione, M. Ackerman, S.L., Letts, V.A. Canti, C., Brodbeck, J., Meir, A., Page, K.M., Kusumi, K., Perez-Reyes, E. Lander, E.S., Frankel, W.N., Gardiner, R.M., Dolphin, A.C. and Rees, M. (2001)
Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.
Journal of Neuroscience 21 6095-6104
Raghib, A., Bertaso, F., Davies A., Page K. M., Meir A. Bogdanov, Y. and Dolphin A. C. (2001)
Dominant negative synthesis suppression of voltage-gated calcium channel CaV2.2 induced by truncated constructs
Journal of Neuroscience 21 8495-8504
Canti, C., Page, K.M., Stephens, G.J. and Dolphin, A.C. (1999)
Identification of residues in the N terminus of α1B critical for inhibition of the voltage-dependent calcium channel by Gβγ
Journal of Neuroscience 19: 6855-6864