Voltage-gated sodium (Nav) channels initiate neuronal and cardiac action potentials. Nav channels co-assemble with other proteins into macromolecular clusters on the neuronal and cardiomyocyte plasma membrane. Yet the wider protein composition of these clusters, the mechanisms by which they are stabilised and their functional significance is poorly understood.
Nav channels contain an alpha subunit containing the ion-selective pore and beta subunits. We have shown that beta3 subunits form trimers on the plasma membrane, and can cross-link individual Nav alpha subunits (1). The project will test the hypothesis that beta3-induced cross-linking helps stabilise both the Nav channels within these plasma membrane clusters, and their broader protein composition. The project will examine the co-localisation of Nav channel alpha subunits with known cluster components such as potassium and calcium channels on neurones and cadiomyocytes from wild –type mice and mice lacking beta3 expression (Scn3b-/- mouse) (2). We will correlate any localisation changes with differences in electrophysiological properties between cells from the wild-type and Scn3b-/- mice.
To provide a more comprehensive inventory of the proteins within these clusters, and their changes in the absence of beta3, we will exploit a new proteomic method that enables the detection of proteins in localised plasma membrane clusters (3). Here, peroxidase is targeted to the Nav channel alpha subunits that biotinylates proteins within a few nanometres from the channel. These proteins are isolated by affinity chromatography and identified by mass spectrometry (3).
This project integrates cell-biological, electrophysiological and proteomic approaches to a major problem in contemporary neurobiology.
1. 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. The Journal of biological chemistry 289, 10797-10811
2.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 physiologica 198, 47-59
3.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. The Journal of biological chemistry 289, 14434-14447