Functional Expression of L-, N-, P/Q-, and R-Type Calcium Channels in the Human NT2-N Cell Line

Abstract

The biophysical and pharmacological properties of voltage-gated calcium channel currents in the human teratocarcinoma cell line NT2-N were studied using the whole cell patch-clamp technique. When held at −80 mV, barium currents ( I Bas) were evoked by voltage commands to above −35 mV that peaked at +5 mV. When holding potentials were reduced to −20 mV or 5 mM barium was substituted for 5 mM calcium, there was a reduction in peak currents and a right shift in the current-voltage curve. A steady-state inactivation curve for I Ba was fit with a Boltzmann curve ( V 1/2 = −43.3 mV; slope = −17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell I Bas were partially blocked by specific channel blockers to a similar extent in 1- to 3-wk and 7- to 9-wk postdifferentiation NT2-N cells: 10 μM nifedipine (19 vs. 25%), 10 μM conotoxin GVIA (27 vs. 25%), 10 μM conotoxin MVIIC (15 vs. 16%), and 1.75 μM SNX-482 (31 vs. 33%). Currents were completely blocked by 300 μM cadmium. In the presence of nifedipine, GVIA, and MVIIC, ∼35% of current remained, which was reduced further by SNX-482 (7–14% of current remained), consistent with functional expression of L-, N-, and P/Q-calcium channel types and one or more R-type channel. The presence of multiple calcium currents in this human neuronal-type cell line provides a potentially useful model for study of the regulation, expression and cellular function of human derived calcium channel currents; in particular the R-type current(s).