TTX-Sensitive and -Resistant Na+ Currents, and mRNA for the TTX-Resistant rH1 Channel, Are Expressed in B104 Neuroblastoma Cells
Gu, Xiang Q., Sulayman Dib-Hajj, Marco A. Rizzo, and Stephen G. Waxman. TTX-sensitive and -resistant Na+ currents, and mRNA for the TTX-resistant rH1 channel, are expressed in B104 neuroblastoma cells. J. Neurophysiol. 77: 236–246, 1997. 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/μ2 (mean ± SE). Time-to-peak (t-peak), activation (τm), and inactivation time constants (τh) for Na+ currents in B104 cells were 1.0 ± 0.04, 0.4 ± 0.06, and0.9 ± 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, τm, and τh 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 α 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 αSNS, 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.