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. Department of Neurology, Yale Medical School, New Haven, CT 06510; Neuroscience Research Center (127A), VA Medical Center, West Haven, CT 06516.
APStracts 3:0206N, 1996.
ABSTRACT
In order to examine the molecular basis for membrane excitability in a neuroblastoma cell line, we used whole-cell patch-clamp methods and RT-PCR to study Na+ currents and channels in B104 cells. We distinguished TTX-sensitive and TTX-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 /[mu]m2. 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 ms, 0.4+/-0.06 ms and 0.9+/-0.04 ms at - 10 mV. The peak conductance-voltage relationship had a V1/2 of -39.8+/-1.5 mV. V1/2 for steady- state inactivation was -81.6+/-1.5 mV. TTX-sensitive and TTX-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 TTX-resistant Na+ currents were kinetically distinct; time-to-peak, [tau]m, and [tau]h for TTX-sensitive currents (0.98+/-0.07 ms; 0.45+/-0.09 ms; 0.64+/-0.31 ms at -10 mV) were shorter than for TTX-resistant currents (1.29+/-0.04 ms; 0.48+/-0.06 ms;1.0+/-0.09 ms at -10 mV). Steady-state voltage dependence of the two currents was indistinguishable. The presence of TTX-sensitive and TTX-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 ([alpha]III, NaG and Na6) 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 TTX-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.

Received 11 July 1996; accepted in final form 9 September 1996.
APS Manuscript Number J538-6.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 7 October 1996