NAVEX

Macromolecular Voltage-Gated Na+ Channel Complexes in the Regulation of Normal and Diseased Cardiac Excitability

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 Obiettivo del progetto (Objective)

'The dynamic modulation of ion channels is crucial to the regulation of cardiac excitability and defects in channel modulation, associated with congenital and acquired cardiac diseases, lead to the development of life threatening arrhythmias. The dynamic modulation of functional expression of ion channels is achieved through changes in cell surface expression and/or functioning of pore-forming channel subunits. Some of the mechanisms used to achieve these regulations involve interactions of pore-forming channel subunits with accessory subunits and post-translational modifications (PTM) of channel components. The voltage-gated Na (Nav) current (INa), generated by Nav1.5 channels, is a key regulator of cardiac excitability, modulating action potential waveforms, refractoriness and propagation. Previous studies have linked family mutations in genes encoding Nav1.5 and Nav1.5 interacting proteins with cardiac arrhythmias, and parallel analyses have suggested roles for these mutations in dysregulating Nav1.5 channel functional expression. However, the mechanisms whereby such native alterations in Nav1.5 function are achieved require identification and functional analysis of in situ Nav1.5 channels. The goal of the present proposal, therefore, is (1) to characterize the native components of Nav1.5 channel complexes, as well as PTM of these components, in the heart, by the use of mass spectrometry analyses; (2) to investigate the role(s) of the previously identified accessory subunit ankyrin G in regulating the functional expression of Nav1.5-encoded INa channels; and (3) to analyze the regulation defects associated with the E1053K mutation in Nav1.5, associated with Brugada syndrome, at the molecular, cellular and whole-animal levels. Altogether, the research proposed will improve our scientific knowledge about ion channel regulation in normal and diseased cardiac excitability, which is needed for improved identification, prevention and treatment of cardiac arrhythmias.'

Introduzione (Teaser)

Cardiac arrhythmias are disorders in the electrical rhythm of the heart, associated with higher risk of sudden cardiac death. EU-funded researchers studied voltage-gated sodium (Nav) channels in native cardiac cells to find solutions.

Descrizione progetto (Article)

Ion channels in the heart such as Nav1.5 are key regulators of cardiac rhythm initiation and propagation. Genetic mutations usually of hereditary nature such as Brugada syndrome cause dysregulation in ion channels resulting in potentially fatal arrhythmias. To identify the molecular and cellular mechanisms involved in regulating electrical excitation in the heart, scientists initiated the NAVEX project.

NAVEX developed and applied a mass spectrometry-based proteomic approach for in situ investigation of Nav channel complexes at the molecular level. For the study, they immunoprecipitated Nav channel complexes and associated regulatory proteins from adult mouse cardiac ventricles. Several novel Nav channel regulatory proteins of were identified. Out of these, the Eps15 interacting protein 2, the Oxysterol-binding protein-Related Protein 11 (ORP11) and the plakoglobin were further analysed. Nav1.5 co-immunoprecipitated with each of these proteins, confirming their association with Nav1.5.

Transient knockdown of ORP11 or plakoglobin expression in rat heart muscle cells did not reveal alterations in Nav1.5 total or cell surface expression. These results revealed the need for assessment of their biophysical properties.

Scientists successfully realised the first ever in situ phosphorylation map of cardiac Nav1.5 channels. They identified eleven serine/threonine phosphorylation sites out of which eight were previously unknown. Ten sites were located in the first intracellular linker loop, indicating the importance of this region in Nav1.5 channel expression and function. Biochemical analyses of phosphomutants revealed two new serine phosphorylation sites involved in regulating Nav1.5 channels' cell surface expression. Findings were published in the Journal of Proteome Research.

The cardiac Nav1.5 channel has proven to be a relatively effective target for anti-arrhythmic drug development. However, these channels had not been characterised until the advent of this project. NAVEX activities could provide the foundation for developing innovative preventive, diagnostic and therapeutic options. Project members also benefit as these findings place them in a competitive position to receive funding for future research projects.