A novel polyclonal antibody specific for the Nav1.5 voltage-gated Na+ channel 'neonatal' splice form

Voltage-gated Na+ channel (VGSC) diversity is achieved through a number of mechanisms: multiple subunits, multiple genes encoding the pore-forming VGSC α-subunit and multiple gene isoforms generated by alternative splicing. A major type of VGSCα alternative splicing is in D1:S3, which has been proposed to be developmentally regulated. We recently reported a D1:S3 spliced form of Nav1.5 in human metastatic breast cancer cells. This novel 'neonatal' isoform differs from the counterpart 'adult' form at seven amino acids (in the extracellular loop between S3-S4 of D1). Here, we generated an anti-peptide polyclonal antibody, named NESOpAb, which specifically recognised 'neonatal' but not 'adult' Nav1.5 when tested on cells specifically over-expressing one or other of these Nav1.5 spliced forms. The antibody was used to investigate developmental expression of 'neonatal' Nav1.5 (nNav1.5) in a range of mouse tissues by immunohistochemistry. Overall, the results were consistent with nNav1.5 protein being more abundantly expressed in selected tissues (particularly heart and brain) from neonate as compared to adult animals. Importantly, NESOpAb blocked functional nNav1.5 ion conductance when applied extracellularly at concentrations as low as 0.05 ng/ml. Possible biological and clinical applications of NESOpAb are discussed.