Structure and function of the sodium channel beta 3-subunit:
In electrically-excitable cells, the voltage-gated sodium (Nav) channel initiates the action potential. Sodium channels are major pharmacological targets and are implicated in pathologies such as heart disease, epilepsy, and chronic pain.
The sodium channel consists of a ~ 260 kDa alpha-subunit with associated ~ 35-40 kDa beta-subunits. The beta-subunits modulate channel electrophysiological behaviour and help traffic the channel to the plasma membrane. There are ten alpha-subunit and four beta-subunit genes, expressed in distinct tissue-specific patterns. All Nav beta-subunits possess a single extracellular immunoglobulin (Ig) domain, connected via a stalk to an alpha-helical transmembrane domain and an intracellular carboxy-terminal region.
We are studying the role played by the beta 3-subunit in Nav channel structure and function. In mice, deletion of the beta 3-subunit gene (Scn3b) is associated with cardiac arrhythmias. To provide a better understanding of the beta 3-subunit, we investigated its structure using X-ray crystallography. We have shown that the beta3-subunits can trimerise via their Ig domains, and induce the formation of Nav channel alpha-subunit oligomers, including trimers. These results provide a new interpretation of previous electrophysiological data, and raise a new set of questions. Does the cross-linking of Nav channels by beta 3 lead to functionally coupled channels? Can different Nav alpha-subunits be cross-linked together? Do the beta-subunits help stabilise the Nav channel into larger-scale protein clusters on the plasma membrane?
Proteomic approaches to plasma-membrane assemblies:
Plasma membrane proteins such as the Nav channels often cluster together selectively as spatially-restricted and functionally integrated complexes. The localised clustering of such proteins can play important roles in many biological processes. Hence the molecular characterisation of surface-bound protein assemblies is of great interest, but represents a major technical challenge. Consequently, there is a need to develop alternative analytical methods that can better survey the diversity of proteins within localised clusters, without assuming direct interaction between near-neighbours.
In collaboration with Sarah Perrett (Institute of Biophysics, Chinese Academy of Sciences, Beijing) and Kathryn Lilley (Cambridge Centre for Proteomics), we have developed a method we call "Selective Proteomic Proximity Labelling Assay Using Tyramide" (SPPLAT). It combines enzyme-labelled antibody targeting with quantitative SILAC proteomics. A peroxidase-conjugated antibody against a protein of interest is allowed to bind cells. Incubation with hydrogen peroxide and a cleavable tyramide-biotin conjugate generates an unstable tyramide free-radical that covalently biotinylates only those proteins in the immediate vicinity of the target. The biotinylated proteins are isolated by streptavidin capture and identified by mass spectrometry. As a proof of principle, SPPLAT has been used to identify proteins co-localised with the cross-linked B-cell receptor (BCR) in the pre-B lymphocyte cell-line DT40. These proteins include integrins, together with novel proteins that we show can regulate integrin activation. We believe that SPPLAT will have wide application to many problems in the biomolecular sciences.
Pance, A., Morrissey-Wettey, F.R., Craig, H, Downing, A, Talbot, R., and Jackson, A.P. (2014) SDF-1 chemokine signalling modulates the apoptotic responses to iron deprivation of clathrin-depleted DT40 cells. PLoS One, e106278.
Li, X. W., Rees, J. S., Xue, P., Zhang, H., Hamaia, S. W., Sanderson, B., Funk, P. E., Farndale, R. W., Lilley, K. S., Perrett, S., and Jackson, A. P. (2014) New Insights into the DT40 B-Cell Receptor Cluster using a Proteomic Proximity Labeling Assay. J Biol Chem 289, 14434-14447
Namadurai, S., Balasuriya, D., Rajappa, R., Wiemhofer, M., Stott, K., Klingauf, J., Edwardson, J. M., Chirgadze, D. Y., and Jackson, A. P. (2014) Crystal Structure and Molecular Imaging of the Nav Channel beta3 Subunit Indicates a Trimeric Assembly. J Biol Chem 289, 10797-10811
Yereddi, N. R., Cusdin, F. S., Namadurai, S., Packman, L. C., Monie, T. P., Slavny, P., Clare, J. J., Powell, A. J., and Jackson, A. P. (2013) The immunoglobulin domain of the sodium channel beta3 subunit contains a surface-localized disulfide bond that is required for homophilic binding. FASEB J 27, 568-580
Cusdin, F. S., Nietlispach, D., Maman, J., Dale, T. J., Powell, A. J., Clare, J. J., and Jackson, A. P. (2010) The sodium channel beta3-subunit induces multiphasic gating in NaV1.3 and affects fast inactivation via distinct intracellular regions. J Biol Chem 285, 33404-33412
Merrick, E. C., Kalmar, C. L., Snyder, S. L., Cusdin, F. S., Yu, E. J., Sando, J. J., Isakson, B. E., Jackson, A. P., and Patel, M. K. (2010) The importance of serine 161 in the sodium channel beta3 subunit for modulation of Na(V)1.2 gating. Pflugers Arch 460, 743-753
Hakim, P., Brice, N., Thresher, R., Lawrence, J., Zhang, Y., Jackson, A. P., Grace, A. A., and Huang, C. L. (2010) Scn3b knockout mice exhibit abnormal sino-atrial and cardiac conduction properties. Acta Physiol (Oxf) 198, 47-59