Cardiac sodium channels expressed in a peripheral neurotumor-derived cell line, RT4-B8.
RT4-B is one of several cell lines derived from a multipotent stem cell line, RT4-AC, which originated from a rat peripheral neurotumor. Based on Northern blot and ribonuclease protection experiments, RT4-B8 cells have been proposed to express rat cardiac Na channel mRNA as the major isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are of the cardiac phenotype with no evidence for subpopulations expressing other Na channel isoforms. Current activation half point (conductance) was -41 +/- 5 mV (n = 7) and the steady-state voltage-dependent availability half point was -89 +/- 1 mV. As expected for cardiac Na channels, the half concentration of block for tetrodotoxin block was 0.74 microM, for saxitoxin (STX) was 0.15 microM, and for the class 2B divalent cation Cd2+ was 67 microM. Block was well described by single-site dose-response relationships with no indication of a subpopulation with "neuronal" affinity. Single-channel conductance (140 mM Na+) was 10 pS and predicted the average number of channels open at peak Na current to be 3 channels/microns2. [3H]STX binding data were also consistent with a single population of low-affinity STX binding sites and predicted channel density to be 11 sites/microns2. No inwardly or outwardly rectifying K or Ca currents were detected electrophysiologically, although in some cells a small time-independent Cl current was detected. Reverse transcription-polymerase chain reaction of mRNA isolated from RT4-B8 cells demonstrated the presence of rat cardiac (rH1) and brain IIa alpha-subunit mRNA, as well as mRNA for the Na channel beta 1-subunit. Northern blot analysis confirmed the predominance of the rat cardiac Na mRNA compared with brain IIa. The beta 1-subunit mRNA levels were significantly lower than those detected in rat brain and heart mRNA but were comparable to the low level of beta 1-subunit mRNA detected in isolated rat ventricular myocytes.
Zeng
D
D
Department of Medicine, University of Chicago, Illinois 60637, USA.
Kyle
J W
JW
Martin
R L
RL
Ambler
K S
KS
Hanck
D A
DA
D000818:Animals; D015152:Blotting, Northern; D004594:Electrophysiology; D009206:Myocardium / Q000378:metabolism*; D010524:Peripheral Nervous System Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D010641:Phenotype; D016133:Polymerase Chain Reaction; D051381:Rats; D012530:Saxitoxin / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D015222:Sodium Channels; D012530:Saxitoxin