A novel polyclonal antibody specific for the Na(v)1.5 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 alpha-subunit and multiple gene isoforms generated by alternative splicing. A major type of VGSCalpha alternative splicing is in D1:S3, which has been proposed to be developmentally regulated. We recently reported a D1:S3 spliced form of Na(v)1.5 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' Na(v)1.5 when tested on cells specifically over-expressing one or other of these Na(v)1.5 spliced forms. The antibody was used to investigate developmental expression of 'neonatal' Na(v)1.5 (nNa(v)1.5) in a range of mouse tissues by immunohistochemistry. Overall, the results were consistent with nNa(v)1.5 protein being more abundantly expressed in selected tissues (particularly heart and brain) from neonate as compared to adult animals. Importantly, NESOpAb blocked functional nNa(v)1.5 ion conductance when applied extracellularly at concentrations as low as 0.05 ng/ml. Possible biological and clinical applications of NESOpAb are discussed.
Chioni
Athina-Myrto
AM
Division of Cell and Molecular Biology, Neuroscience Solutions to Cancer Research Group, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
Fraser
Scott P
SP
Pani
Filippo
F
Foran
Patrick
P
Wilkin
Graham P
GP
Diss
James K J
JK
Djamgoz
Mustafa B A
MB
D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular; D004558:Electric Stimulation / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D016296:Mutagenesis / Q000502:physiology; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques / Q000379:methods; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology; D012333:RNA, Messenger / Q000096:biosynthesis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D014162:Transfection / Q000379:methods
D000906:Antibodies; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D020033:Protein Isoforms; D012333:RNA, Messenger; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; D015222:Sodium Channels