TTX-sensitive and -resistant Na+ currents, and mRNA for the TTX-resistant rH1 channel, are expressed in B104 neuroblastoma cells.
To examine the molecular basis for membrane excitability in a neuroblastoma cell line, we used whole cell patch-clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) to study Na+ currents and channels in B104 cells. We distinguished Tetrodotoxin (TTX)-sensitive and -resistant Na+ currents and detected the mRNA for the cardiac rH1 channel in B104 cells. Na+ currents could be recorded in 65% of cells. In the absence of TTX, mean peak Na+ current density was 126 +/- 19 pA/pF, corresponding to a channel density of 2.7 +/- 0.4/micron 2 (mean +/- SE). Time-to-peak (t-peak), activation (tau m), and inactivation time constants (tau h) for Na+ currents in B104 cells were 1.0 +/- 0.04, 0.4 +/- 0.06, and 0.9 +/- 0.04 ms at -10 mV. The peak conductance-voltage relationship had a V 1/2 of -39.8 +/- 1.5 mV. V 1/2 for steady-state inactivation was -81.6 +/- 1.5 mV. TTX-sensitive and -resistant components of the Na current had half-maximal inhibitions (IC50), respectively, of 1.2 nM and, minimally, 575.5 nM. The TTX-sensitive and -resistant Na+ currents were kinetically distinct; time-to-peak, tau m, and tau h for TTX-sensitive currents were shorter than for TTX-resistant currents. Steady-state voltage dependence of the two currents was indistinguishable. The presence of TTX-sensitive and -resistant Na+ currents, which are pharmacologically and kinetically distinct, led us to search for mRNAs known to be associated with TTX-resistant channels, in addition to the alpha subunit mRNAs, which have previously been shown to be expressed in these cells. Using RT-PCR and restriction enzyme mapping, we were unable to detect alpha SNS, but detected mRNA for rH1, which is known to encode a TTX-resistant channel, in B104 cells. B104 neuroblastoma cells thus express TTX-sensitive and -resistant Na+ currents. These appear to be encoded by neuronal-type and cardiac Na+ channel mRNAs including the RH1 transcript. This cell line may be useful for studies on the rH1 channel, which is known to be mutated in the long-QT syndrome.
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Department of Neurology, Yale Medical School, New Haven, Connecticut 06510, USA.
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Rizzo
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Waxman
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D001932:Brain Neoplasms / Q000378:metabolism*; D004594:Electrophysiology; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011994:Recombinant Proteins; D015222:Sodium Channels; D013779:Tetrodotoxin; D012194:RNA-Directed DNA Polymerase