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 cel l line, RT4-AC, which originated from a rat peripheral neurotumor. Bas ed on Northern blot and ribonuclease protection experiments, RT4-B8 ce lls have been proposed to express rat cardiac Na channel mRNA as the m ajor isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are o f 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 avai lability half point was -89 +/- 1 mV. As expected for cardiac Na chann els, the half concentration of block for tetrodotoxin block was 0.74 m u M, for saxitoxin (STX) was 0.15 mu M, and for the class 2B divalent cation Cd2+ was 67 mu M. Block was well described by single-site dose- response relationships with no indication of a subpopulation with ''ne uronal'' affinity. Single-channel conductance (140 mM Na+) was 10 pS a nd predicted the average number of channels open at peak Na current to be 3 channels/mu m(2). [H-3]STX binding data were also consistent wit h a single population of low-affinity STX binding sites and predicted channel density to be 11 sites/mu m(2). No inwardly or outwardly recti fying K or Ca currents were detected electrophysiologically, although in some cells a small time-independent Cl current was detected. Revers e transcription-polymerase chain reaction of mRNA isolated from RT4-B8 cells demonstrated the presence of rat cardiac (rH1) and brain II alp ha alpha-subunit mRNA, as well as mRNA for the Na channel beta(1)-subu nit. Northern blot analysis confirmed the predominance of the rat card iac Na mRNA compared with brain IIa. The beta(1)-subunit mRNA levels w ere significantly lower than those detected in rat brain and heart mRN A but were comparable to the low level of beta(1)-subunit mRNA detecte d in isolated rat ventricular myocytes.