TTX-SENSITIVE AND TTX-RESISTANT NA-RNA FOR THE TTX-RESISTANT RH1 CHANNEL, ARE EXPRESSED IN B104 NEUROBLASTOMA-CELLS( CURRENTS, AND MESSENGER)

To examine the molecular basis for membrane excitability in a neurobla stoma cell Line, we used whole cell patch-clamp methods and reverse tr anscription-polymerase chain reaction (RT-PCR) to study Na+ currents a nd channels in B104 cells. We distinguished Tetrodotoxin (TTX)-sensiti ve and -resistant Na+ currents and detected the mRNA for the cardiac r H1 channel in B104 cells. Na+ currents could be recorded in 65% of cel ls. In the absence of TTX, mean peak Na+ current density was 126 +/- 1 9 pA/pF, corresponding to a channel density of 2.7 +/- 0.4/mu(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 stead y-state inactivation was -81.6 +/- 1.5 mV. TTX-sensitive and -resistan t components of the Na current had half-maximal inhibitions (IC50), re spectively, 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-resi stant currents. Steady-state voltage dependence of the two currents wa s indistinguishable. The presence of TTX-sensitive and -resistant Nacurrents, which are pharmacologically and kinetically distinct, led us to search for mRNAs known to be associated with TTX-resistant channel s, in addition to the alpha subunit mRNAs, which have previously been shown to be expressed in these cells. Using RT-PCR and restriction enz yme mapping, we were unable to detect alpha SNS, but detected mRNA for rH1, which is known to encode a ?TX-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 us eful for studies on the rH1 channel, which is known to be mutated in t he long-QT syndrome.