Biochemical and Biophysical Characterization of a Sodium Channel Pore Protein

Voltage gated sodium channels (NaVs) are large polytopic membrane proteins involved in the generation of action potentials in excitable cells. Mutations in these channels are involved in arrhythmias, pain disorders, and some forms of hereditary epilepsy. Recently, single subunit NaV homologs have been identified in a large variety of marine bacteria. Sequence conservation at key points along the ion conducting path between the eukaryotic and prokaryotic NaVs suggests that the prokaryotic channels can serve as an excellent model for understanding the basic aspects of NaVs function and the effects of disease mutations that lead to channel malfunction.Using a protein engineering approach based on the likelihood that the voltage-sensor and pore domains are independently folded domains, we dissected the voltage sensor from the ion conducting part of bacterial ion channels to create a stand-alone pore protein. The ‘NaV pore’ expresses a stable protein that can be isolated and purified to homogeneity. Extensive biochemical and biophysical characterization demonstrate that the ‘NaV pore’ self-assembles as tetramers that show the characteristics of an alpha-helical membrane protein. Furthermore, the ‘NaV pore’ conducts sodium currents when incorporated into lipid vesicles. Thus, the ‘NaV pore’ represents an active ion channel that provides an excellent model system for the study of the factors that govern sodium selectivity and permeability.