Electrophysiological Characteristics of Neonatal Nav1.5 Expressed in a Highly Invasive Human Breast Cancer Cell Line: Sensitivity to pH and Divalent Cations

Abstract

Electrophysiological recordings from human carcinoma cell lines have shown consistently that strongly metastatic cells express functional voltage-gated sodium channels (VGSCs). The predominant VGSC in metastatic breast cancer, in vitro and in vivo, is the ‘neonatal’ splice form of Nav1.5. In this developmentally regulated D1:S3 splice variant of Nav1.5, there are 31 nucleotide differences between the 5’-exon (‘neonatal’) and the 3’-exon (‘adult’) forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, a conserved negative aspartate residue in the ‘adult’ is replaced with a positive lysine. ‘Neonatal’ and ‘adult’ Nav1.5 α-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties were investigated by whole-cell patch-clamp recording. Compared with the ‘adult’ isoform, the ‘neonatal’ channel exhibited (1) depolarized threshold of activation and voltage at which current peaked; (2) much slower kinetics of activation and inactivation; (3) ∼50% greater transient charge (Na+) influx; (4) a slower recovery from inactivation; and (5) larger persistent Na+ currents. Mutating the lysine in the ‘neonatal’ channel back to aspartate resulted in the electrophysiological parameters studied reverting strongly back towards the ‘adult’, i.e. the lysine residue was primarily responsible for the electrophysiological differences. The charge difference between the two Nav1.5 isoforms was ‘challenged’ by H+ and Cd2+. The main differential effect occurred at pH 5.25-5.75 in which the activation parameters of ‘neonatal’ Nav1.5 were affected significantly less. The biophysical characteristics of ‘neonatal’ Nav1.5 observed could have significant developmental and pathophysiological consequences. In particular, the prolonged Na+ influx can alter intracellular Ca2+ and/or pH homeostasis, at least in microdomains, and channel activation remains relatively efficient under extreme acidosis.