Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue.
In developmentally regulated D1:S3 splicing 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, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.
Onkal
Rustem
R
Division of Cell & Molecular Biology, Neuroscience Solutions to Cancer Research Group, Sir Alexander Fleming Building, South Kensington Campus, Imperial College London, London, UK.
Mattis
Joanna H
JH
Fraser
Scott P
SP
Diss
James K J
JK
Shao
Dongmin
D
Okuse
Kenji
K
Djamgoz
Mustafa B A
MB
D000328:Adult; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D002460:Cell Line; D005091:Exons; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*
D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D020033:Protein Isoforms; C568320:SCN5A protein, human; D015222:Sodium Channels; D008239:Lysine