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

Torben R. Neelands,1 Anthony P. J. King,2 and Robert L. Macdonald1,2,3

 1Neuroscience Program,  2Department of Neurology, and  3Department of Physiology, University of Michigan, Ann Arbor, Michigan 48104-1687


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neelands, Torben R., Anthony P. J. King, and Robert L. Macdonald. Functional Expression of L-, N-, P/Q-, and R-Type Calcium Channels in the Human NT2-N Cell Line. J. Neurophysiol. 84: 2933-2944, 2000. 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 (IBas) 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 IBa was fit with a Boltzmann curve (V1/2 = -43.3 mV; slope -17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell IBas 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).


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INTRODUCTION
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Prolonged retinoic acid treatment of the human NT2 teratocarcinoma cells induces them to differentiate into a neuron-like phenotype (NT2-N cells) that is morphologically similar to mouse cortical neurons in cell culture (Pleasure et al. 1992). These cells express neuron specific markers on their cell surface, neuronal cytoskeletal markers, and neuronal secretory markers only after differentiation (Pleasure et al. 1992). NT2-N cells express amyloid precursor protein and secrete beta amyloid peptides; these have made this line an important cellular model system in Alzheimer's disease research (Wertkin et al. 1993). NT2-N cells also express muscarinic cholinergic and opioid receptors (Pleasure and Lee 1993; Pleasure et al. 1992); functional ligand-gated receptor ion channels, including GABAA (Matsuoka et al. 1997; Neelands et al. 1998), N-methyl-D-aspartate (NMDA), and kainate receptor channels (Itoh et al. 1998; Matsuoka et al. 1997; Paterlini et al. 1998; Rootwelt et al. 1998); and voltage-gated sodium channels (Greenfield et al. 1997; Matsouka et al. 1997).

In addition to being a valuable model system for human neuronal function, NT2-N cells also show promise as a potential alternative to human embryonic neurons for transplantation into patients with Parkinson's disease (Willing et al. 1999) and possibly other degenerative diseases such as Huntington's disease (Hurlbert et al. 1999). Because NT2-N cells can be transfected, they are also potential vectors for neuronal gene therapy (Trojanowski et al. 1997). Important for understanding whether these cells can operate as functional neurons is whether they possess normal neuronal mechanisms for calcium-dependent neurotransmitter release and synaptic neurotransmission. Functional synapses have been shown to form between NT2-N cells in culture when they are grown on astrocytes (Hartley et al. 1999). While there has been a report of voltage-dependent calcium currents in NT2-N cells (Sanderson et al. 1997) and of potentially channel-facilitated cytosolic calcium transients (Gao et al. 1998a), NT2-N cell calcium channels have not been characterized.

Voltage-gated calcium channels have been classified based on their pharmacological and biophysical characteristics (Fox et al. 1987; for recent reviews, see: Jones 1998; Moreno 1999). Low-voltage-activated (LVA) T-type currents are distinguished from high-voltage-activated (HVA) L-, N-, P-, Q-, and R-type currents based on the relatively negative potentials at which they are activated (Fox et al. 1987). Biophysical properties, such as activation and inactivation kinetics, have also been used to classify calcium currents. T-type currents typically activate slowly and inactivate rapidly (Fox et al. 1987; Perez-Reyes et al. 1998). HVA currents can be separated based on their sensitivity to specific blockers and toxins: L-type channels are sensitive to dihydropyridines (Tsien et al. 1988), N-type channels are irreversibly blocked by the snail toxin omega -CTx-GVIA (Reynolds et al. 1986), and N-, P-, and Q-type currents are all reversibly inhibited by omega -CTx-MVIIC (Hillyard et al. 1992). Preferential block by the spider toxin omega -agatoxin-IVA can be used to isolate P-type channels (Mintz et al. 1992). Residual currents insensitive to all of these compounds ("R-type" current) have suggested the presence of other channels. A recently developed R-type peptide inhibitor SNX-482 specifically and nearly completely blocked the "resistant" current from acutely dissociated neurohypophyseal nerve endings, suggesting that it may be a specific blocker for R-type currents (Newcomb et al. 1998). However, this compound did not block toxin-resistant currents in other neurons, including those from cerebellar granule cells, retinal ganglion cells, or hippocampal pyramidal cells (Newcomb et al. 1998), suggesting that multiple calcium current subtypes may comprise the R-type current. Furthermore a recent study indicates the presence of three distinct components of R-type current, with differential sensitivity to SNX-482, in rat cerebellar granule cells (Tottene et al. 2000).

Through the use of molecular cloning, a number of subunits have been described, consistent with the diversity in calcium channel currents. Five neuronal alpha 1 subunits (A-E) have been described that form the channel pore (for review, see: Hofmann et al. 1994; McCleskey 1994; Perez-Reyes and Schneider 1995). Expression studies demonstrated that the pharmacological properties of alpha 1 subunit currents were similar to those of native calcium channel type currents (L-type: alpha 1C, alpha 1D; N-type: alpha 1B; Q/P-type: alpha 1A; R-type: alpha 1E) (McCleskey 1994; Zhang et al. 1993). Identification of the calcium channel cDNA responsible for specific functional calcium current types is incomplete, however, possibly due to the co-ordinate expression of auxiliary subunits (Moreno et al. 1997). The subunit composition of native calcium channels is further complicated by a diversity of auxiliary subunits including alpha 2(A-E)/delta , beta 1-4, and gamma  subunits. Differential regional expression and cellular localization of calcium channel types have been well documented and could be important in controlling calcium-dependent cellular processes such as neurotransmitter release, gene expression, and neurite outgrowth.

Recent studies in NT2-N cells have demonstrated expression of calcium channel subunits alpha 1B (N type) and alpha 1D (L type) mRNA and cytosolic calcium transients that were blocked by nifedipine and omega -conotoxin GVIA (Gao et al. 1998a), consistent with the functional expression of N- and L-type calcium channels. Differentiated NT2-N cells are a relatively homogenous population of human neurons that can be grown in large quantities and maintained under laboratory conditions. Therefore the NT2-N cell line may be useful in molecular and electrophysiological studies on the functional role, localization, and composition of different human calcium channel types and their regulation by neuronal activity and various neurotransmitters and neuromodulators. In this study, we describe the isolation of at least four different HVA calcium channel types (L-, N-, P/Q-, and R-) in NT2-N cells and the absence of a significant LVA T-type current.


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Cell culture

Cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) HG with 10% fetal bovine serum and penicillin/streptomycin added as previously described (Pleasure et al. 1992). Cells were plated at 2 × 106 cells/75 cm2 flask and differentiated by treatment with 1 µM retinoic acid (RA) for 4 wk. After RA treatment, cells were washed with versene then treated with trypsin to dislodge the cells. Cells were resuspended after being triturated and replated at a 1:10 dilution with DMEM HG and maintained in 5% CO2. The following day the media was removed and saved as conditioned media to feed replate 2 and 3. Cells were again treated with trypsin and centrifuged for 5 min at 1,000 rpm. The pellet was resuspended in 1 ml of media containing mitotic inhibitors (10 µM fluorodeoxyuridine + 10 µM uridine and 1 µM cytosine arabinoside) and replated (replate 2). The same treatment was performed again after 1 wk in culture (replate 3) to obtain nearly 100% pure, terminally differentiated neuron-like cells. These cells were replated onto 35 mm Corning dishes coated with 10 ng/ml poly-D-lysine and Matrigel (Collaborative Biomedical, Bedford MA), a solubilized basement membrane extracted from the Engelbreth-Holm-Swarm mouse sarcoma (Kleinman et al. 1982), for electrophysiological testing. Cells were recorded from either 1-2 or 8-10 wk after final plating.

Solutions and drug application

Cells were removed from 5% CO2 incubator and the medium was replaced with a barium external solution [(in mM) 106 NaCl, 5 BaCl2, 2 MgCl2, 2.5 KCl, 10 dextrose, 3 CsCl, 25 TEA, 0.1 3,4-diamino-pyridine, and 20 HEPES and 5 µM TTX] with a pH of 7.35, 295-305 mosM. Patch-clamp electrodes of 5-10 MOmega were filled with internal solution [(in mM) 95 cesium acetate, 35 CsCl, 10 HEPES, 10 EGTA, 3 TEA, 5 ATP, and 0.3 GTP] with a pH of 7.25, 275-285 mosM. Stock solutions of omega -conotoxin-GVIA (omega -CTx GVIA) were dissolved in sterile water and frozen in 500-µl aliquots. Stock solutions of nifedipine were dissolved in dimethylsulfoxide (DMSO) and diluted with sterile water (final DMSO concentration was less than 1:1000). Individual drugs were diluted in external solution to their final concentration, and tubes containing nifedipine were wrapped in aluminum foil. omega -CTx-GVIA and omega -conotoxin-MVIIC (omega -CTx-MVIIC) were purchased from RBI, Natick, MA. SNX-482 was obtained as a kind gift of Dr. George Miljanic of Neurex, division of Elan Pharmaceuticals. Tetrodotoxin (TTX) was purchased from Calbiochem, La Jolla, CA. 3,4-Diaminopyridine was purchased from Aldrich Chemical, Milwaukee, WI. All other compounds were purchased from Sigma Chemical, St. Louis, MO. Compounds were applied to cells using either a modified U-tube application system (Greenfield and Macdonald 1996) or through pressure ejection or large aperture (ca. 50 µm) "weeper" pipettes. The U-tube system enabled us to position a micropipette with a 40- to 50-µm tip next to the cell for the duration of the recording and successively apply multiple drugs to each cell. The pressure ejection and weeper pipettes each contained a single concentration of drug and were positioned ~100 µm from the cell. The weeper pipette was a capillary tube with a low-resistance tip that allowed the drug to simply flow out of the pipette without any back pressure.

Electrophysiology

Whole cell voltage-clamp recordings using the patch-clamp technique were obtained as described previously (Hamill et al. 1981) using an Axopatch 1-B or 1-D amplifier (Axon Instruments). Patch-clamp electrodes were pulled from micro-hematocrit capillary tubes (Labcraft) using a P-87 Flaming-Brown micropipette puller (Sutter Instrument Co.). Signals were low-passed filtered at 2 kHz using an eight-pole Bessel filter then digitized at 2 kHz, recorded and analyzed using pClamp6 or pClamp7 software (Axon Instruments). All currents were leak subtracted using the P/4 protocol provided in the software package. Data were analyzed, Boltzmann and dose response curves were generated and figures created off-line using GraphPad Prism software (GraphPad Software). All data are presented as means ± SE.


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NT2-N cells

NT2 cells (Fig. 1A) were grown to confluence and treated with 1 µM retinoic acid for 5 wk and then plated onto Matrigel extracellular matrix in the presence of mitotic inhibitors from 1 to 9 wk. Cells demonstrated a neuron-like morphology (Fig. 1B) within 1-2 days, and cells with a pyramidal-like morphology and multiple dendritic and axonal processes were selected for study. The majority of the cells observed had two or three neurites. Cells began to assemble into clusters of 20 to >200 cells, and to pile on top of one another after several weeks of being plated on matrix (Fig. 1C). Long processes resembling bundles of axons extended among the clusters. For this study, only visibly isolated, nonpiled cells with pyramidal morphology and outside of the clusters were selected.



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Fig. 1. Photomicrographs of NT2-N cells. A: NT2 "stem" cells before differentiation protocol. B: NT2-N cells after 5-wk retinoic acid differentiation protocol and plated onto plates covered with Matrigel protein matrix, illustrating the pyramidal shaped multiple processed morphology chosen for this study. C: NT2-N cells 9 wk after retinoic acid differentiation protocol.

NT2-N cells express voltage-dependent calcium channel currents

Voltage-dependent calcium channel currents were studied using barium (5 mM) as the charge carrier. Maximal inward barium currents (IBas) were evoked by step commands to +5 mV from a holding potential (Vh) of -80 mV (see METHODS) with maximal leak-subtracted peaks ranging from <0.100 to >1500 pA in 1- to 10-wk-old NT2-N cells. The quality of the seals obtained was judged by apparent whole cell leak currents and duration of tail currents when depolarizing steps were made from Vh = -80 mV, and cells were excluded if estimated tail current tau deacativation was >10 ms. Although the majority of cells selected for recording extended two to three neurites, the quality of the voltage clamps obtained, as judged by estimated tau deactivation of IBa, appeared to be good. Cells showing evidence of poor voltage clamp (e.g., IBa evoked by step to +5 mV from Vh = -80 mV tau deactivation >10 ms) were excluded from analysis. Figure 2 shows analyses of the voltage-dependent IBa current-voltage relationships for NT2-N cells at 1-2 and 7-10 wk in culture postdifferentiation. A step protocol that delivered a series of 100-ms voltage steps ranging from -110 to +90 mV from a holding potential of -80 mV, designed to define the range of activation at 5-mV increments and to determine the reversal potential with either 10- or 15-mV step intervals, was used to determine current-voltage relationships. Superimposed current traces evoked from NT2-N cells (Vh = -80 mV) by steps -110 mV through +5 mV are shown in Fig. 2, A (1 wk in culture) and B (8 wk in culture). Figure 2, C and D, shows peak current-voltage relationships with incremental steps made from -110 to +95 mV from NT2-N cells 1-2 wk (n = 77) and from -110 to +110 mV from NT2-N cells 8-10 wk (n = 17) in culture, respectively. IBas were activated positive to -35 mV, peaked at +5 mV, and reversed around +90 mV in both groups of NT2-N cells, typical of high-threshold calcium channel currents. When the linear portions of the falling phase of the current-voltage relationships were fit with linear regressions, the resulting fitted lines crossed the abscissa between +60 and +70 mV, consistent with the Nernst equation. Figure 2E shows conductance-voltage curves calculated from these data using +60 mV as the reversal potential and fit with Boltzmann curves, with half activation voltages (V1/2) of -4.5 and -7.8 mV and slopes of 7.3 and 5.9 mV/pA for 1- to 2-wk-old and 8- to 10-wk-old NT2-N cells, respectively.



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Fig. 2. Voltage-dependent IBas evoked from 2- and 9-wk postdifferentiation NT2-N cells. Calcium channel currents were recorded in the whole cell patch-clamp configuration using barium (5 mM) as the charge carrier (see METHODS). Currents were evoked from a holding potential (Vh) of -80 mV by 300-ms step commands to -110 through +95 mV from 2-wk postdifferentiation and to -110 through +110 mV from 9-wk postdifferentiation NT2-N cells at 1-min intervals and leak subtracted using a P/4 protocol. A-D: IBa s evoked from NT2-N cells at 1-2 wk (A and C) and 8-10 wk (B and D) after differentiation. Voltage steps evoked inward IBas beginning at about -30 mV. A and B show superimposed representative current traces from 1- to 2- and 8- to 10-wk postdifferentiation NT2-N neurons evoked by voltage steps from Vh = -80 to +95 or +110 mV, respectively. C and D show current-voltage relation of averaged peak currents evoked from 1- to 2-wk (C, n = 77) and 8- to 10-wk (D, n = 17) NT2-N cells, from a holding potential of -80 mV. Calcium channel currents were activated at about -30 mV, peaked at +5 mV and reversed around +90 mV. E: conductance-voltage relation of the data shown in Fig. 3, C and D, using the Nernst equation predicted reversal potential of +60 mV. , 1- to 2-wk postdifferentiation; black-triangle, 8- to 10-wk postdifferentiation. Inset: data normalized to peak current. F: peak current amplitude compared with time in culture of NT2-N cells postdifferentiation. G: rundown of IBa in NT2-N cells. Peak IBa were measured over a 30-min time period and normalized to the maximum current recorded in that cell. Currents increased in amplitude by 10-40% for the first 5-7 min then slowly decreased back to initial levels. H: representative superimposed current traces from a 9-wk postdifferentiation NT2-N cell evoked at 5 and 30 min after patch rupture.

The maximal peak current amplitude increased substantially from 1-wk [232 ± 135 (SE) pA] to 10-wk (1025 ± 130 pA) postdifferentiation (Fig. 2F). To determine time-dependent effects on IBa amplitude and current "rundown," NT2-N cells were stepped from -80 to +5 mV (peak of current-voltage relation) at 1-min intervals for 30 min (n = 5/6; Fig. 2G). Peak amplitudes of whole cell currents typically increased ~20-40% during the first 5 min of recording and then slowly decreased back to initial levels (Fig. 2H). As there was little change in the peak current over this recording time, no correction was made for rundown in subsequent experiments. Individual whole cell currents from all experiments varied in the extent of inactivation that occurred during a 300-ms step to the maximal current. There was no apparent correlation between cellular morphology and the amount of inactivation of the whole cell current (data not shown).

Figure 3 shows the effects of holding potential and of changing charge carrier on the current-voltage and conductance-voltage relations in NT2-N cells in 1- to 2-wk postdifferentiation NT2-N cells (n = 4). Maximal peak current at all step potentials was achieved when cells were held at -80 mV or higher (Fig. 3A, n = 4 cells). As the holding potential was made less negative, the calculated V1/2 was shifted to the right (Fig. 3A), and the conductance decreased in a nonlinear fashion (Fig. 3B) similar to the steady-state inactivation curves described in the following text. It is possible that this right-shift at reduced holding potentials may in part reflect improved voltage clamp at these potentials. When cells were held at -100 mV, the current-voltage relations were similar to those produced when the cells were held at -80 mV, providing no indication that low-threshold T-type calcium currents were activated (data not shown). Steady-state inactivation curves were generated by stepping from varying holding potentials (-100 to 0 mV) to the peak of the current-voltage curve (+5 mV) in 1- to 3-wk-old NT2-N cells. Averaged normalized data were fit with a Boltzmann curve with a V1/2 of -43.3 mV and a slope of -17.7 mV (n = 17; Fig. 3C). Fits of the steady-state inactivation curves from individual cells had similar values for these parameters when averaged (V1/2 = -42.6 mV, slope = -15.5 mV). The calculated V1/2 and slope of Boltzmann curves fitted to data obtained from individual cells were evenly distributed around the mean values (Fig. 3C, inset). The variation of the V1/2 may represent the cell-to-cell variation in the proportion of different types of calcium channel or may also reflect small differences in voltage clamp between cells with varying length or number of neurites. Repeating these protocols in 8-wk-old NT2-N produced similar results (data not shown).



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Fig. 3. Effects of holding potential and charge carrier on the current-voltage relation and steady-state inactivation of IBa evoked from NT2-N cells. A: current-voltage relations of 1- to 2-wk postdifferentiation NT2-N neurons with varying holding potentials from -80 to -20 mV (n = 5). Averaged peak currents were plotted against the step potential for each holding potential. The maximal current was reduced nonlinearly as the holding potential was made more positive. B: conductance-voltage relations with varying holding potentials were calculated from data in Fig. 3A. Peak conductance was decreased and the voltage of half activation (V1/2) became more positive as the holding potential became more positive. C: currents were evoked from 1- to 2-wk postdifferentiation NT2-N neurons by step commands from various holding potentials to +5 mV. Normalized peak currents are plotted as a function of holding potential and fit with a Boltzmann sigmoidal curve with slope of -17.7 mV and voltage of half activation of -43.3 mV (n = 17). Individual slopes and voltages of half activation were evenly distributed around the mean (inset; horizontal bar = mean). D: current-voltage relationship of whole cell currents evoked from NT2-N cells (Vh = -80 mV) with 5 mM barium () or 5 mM calcium (black-triangle) as the charge carrier. Averaged peak currents were slightly reduced over the range of step potentials and maximal responses were shifted to the right with 5 mM calcium in the external solution. E: conductance-voltage relations using the same data as in D. A rightward shift in the voltage of half activation from -7.2 to +0.8 mV occurred when calcium was substituted for barium.

The effects of the charge carrier on the voltage-dependent properties of whole cell calcium channel currents in 1- to 2-wk-old NT2-N cells was investigated by replacing 5 mM barium with 5 mM calcium in the recording solution. Average maximal peak currents were slightly reduced from 317 ± 60 pA when barium was the charge carrier (n = 5 cells) to 263 ± 52 pA when calcium was the charge carrier (n = 4 cells, Fig. 3D). The peak of the current-voltage relation increased from +5 mV in barium containing external solution to +15 mV when calcium was the charge carrier. Boltzmann fits of averaged conductance-voltage relations under these two conditions did not show a significant difference in the peak conductance or slope. There was, however, a change in the V1/2 from -7.2 mV in barium to +0.9 mV in calcium with no alteration in the slope (Fig. 3E).

Effects of L-, N-, and P/Q-type specific channel blockers

The percent contribution of various calcium channel types to the total whole cell current in NT2-N cells of various ages (1- to 3- vs. 7- to 10-wk-old NT2-N) was investigated by using specific blockers of the different channel types. Whole cell currents were evoked at 1-min intervals by stepping to +5 mV from a holding potential of -80 mV for 300 ms. Individual voltage step commands were then repeated until the peak current had stabilized. Voltage commands were then repeated in the presence of omega -CTx GVIA, nifedipine, omega -CTx MVIIC, and/or cadmium applied with pressure ejection (omega -CTx GVIA), weeper pipettes (omega -CTx MVIIC), or a modified U-tube (nifedipine and cadmium) (Greenfield and Macdonald 1996) positioned near the cell. When multiple channel blockers were applied, the irreversible N-type calcium channel toxin omega -CTx GVIA (10 µM) was applied first and then removed from the recording media to spare P/Q currents. omega -CTx MVIIC (10 µM), a toxin that blocks N-, P-, and Q-type calcium channels, was applied following treatment by omega -CTx GVIA, thus making it specific for P/Q-type channels. In the 1-wk postdifferentiation NT2-N cell analyzed in Fig. 4A, omega -CTx GVIA (10 µM) reduced peak IBa from 277 to 128 pA (46% inhibition). The pipette was then removed from the recording solution, and peak currents from subsequent voltage commands did not recover (Fig. 4A). Nifedipine (10 µM) was then applied, which further reduced the current to 84 pA (an inhibition of 16% of the original current and 35% of the residual CTx-GVIA-insensitive current). Current amplitude recovered after the pipette containing nifedipine was removed. The cell was then treated with nifedipine (10 µM) and omega -CTx MVIIC (10 µM), which reduced the current to 58 pA. This further reduction (representing ~10% of the original current) from nifedipine alone thus appeared to be due to P/Q-type currents. The residual "toxin-resistant" current represented 21% of the original current in this cell.



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Fig. 4. Effects of blockers of L-, N-, and P/Q-type calcium channels on NT2-N cell IBa. Sensitivity to subtype-specific calcium channel blockers of whole cell IBa evoked from NT2-N cells by 300-ms steps from a holding potential of -80 to +5 mV was measured. A: peak current amplitude vs. time from a 1-wk postdifferentiation NT2-N cell that was treated sequentially with omega -conotoxin GVIA (omega -CTx-GVIA, 10 µM), nifedipine (10 µM), and a combination of nifedipine (10 µM) and omega -conotoxin MVIIC (omega -CTx-MVIIC, 10 µM) at the times indicated (). B: current traces showing the irreversible block of the control current (1) by omega -CTx GVIA (10 µM) (2), followed by the block by nifedipine (10 µM) (3), the subsequent recovery from inhibition by nifedipine (4), and the complete block by cadmium (300 µM) (5). C: relative contribution of the different channel types to the whole cell current determined by the percentage of the total peak IBa blocked by omega -CTx GVIA (10 µM); nifedipine (10 µM); or omega -CTx-MVIIC (10 µM) after previous omega -CTx GVIA treatment, in 1- to 3-wk () and 7- to 10-wk () postdifferentiation NT2-N cells. A significant proportion of current remained after co-application of all 3 inhibitors. D: the percentage of the total IBa blocked in individual NT2-N cells by each inhibitor was evenly distributed around the mean (horizontal bar).

Nifedipine inhibited approximately the same percentage of the original peak current whether applied to untreated cells or to cells previously treated with CTx-GVIA, and currents quickly recovered after nifedipine was removed. Figure 4B shows currents from an NT2-N cell (1-wk postdifferentiation) evoked by steps to +5 mV before (trace 1) and after (trace 2) treatment with CTx-GVIA (10 µM), which reduced peak current by 21%. Subsequent application of nifedipine (10 µM) reduced current by a further 30% (trace 3), but current quickly recovered after nifedipine was removed (trace 4). Cadmium (300 µM) completely blocked IBa (trace 5).

omega -CTx GVIA (10 µM) reduced peak currents by ~30% in both 1- to 2- and 8- to 10-wk-old NT2-N cells (Fig. 4, C and D). omega -CTx MVIIC (10 µM) on average blocked ~15% of the total current in all ages of NT2-N cells when applied after previous treatment with omega -CTx GVIA (Fig. 4, C and D). Nifedipine (10 µM) inhibited a slightly larger proportion of the IBa on average in 8- to 10-wk-old NT2-N cells (25 ± 7%, n = 18) compared with 1- to 2-wk-old NT2-N cells (19 ± 1.2%, n = 30). Application of the calcium channel antagonist cadmium (300 µM) totally blocked the current evoked by step commands in all cells tested (Fig. 4B, trace 5). The percentage of total current blocked in individual cells by each channel blocker was evenly distributed around the mean (Fig. 4D). A significant proportion (an average of 37.5% in 1- to 2-wk-old and 33% in 8- to 10-wk-old NT2-N) of total current still remained after co-application of all three blockers (Fig. 4C). This residual toxin-resistant current suggests the presence of channel-type(s) in addition to the L-, N-, P-, and Q-type channels, possibly including channels producing R-type currents.

Effects of SNX-482 on NT2-N calcium channel currents

To further characterize the toxin-resistant currents in NT2-N cells, we tested the sensitivity of total and toxin-resistant calcium channel currents to a newly developed peptide channel blocker, SNX-482. This peptide channel blocker specifically blocks currents from recombinant E-class calcium channels and the toxin-resistant (R-type) currents in neurohypophyseal neurons (Newcomb et al. 1998). We first examined the effects of SNX-482 on total NT2-N IBa. Figure 5A shows IBa traces evoked by a step to +5 mV from a 7-wk postdifferentiation NT2-N cell after treatment with increasing concentrations of SNX-482. In this cell, SNX-482 (1.1 µM) decreased maximal whole cell IBa by 34% and decreased apparent inactivation rate but did not decrease apparent activation rate, consistent with the block of an inactivating current component. A maximal inhibition of ~38% of total IBa and an IC50 of 119 nM was derived from a sigmoidal concentration-response curve of current inhibition fit to pooled data from several cells treated with SNX-482 (1 nM to 5 µM, Fig. 5B). Treatment of cells with concentrations of SNX-482 >10 µM led to inhibition of a greater proportion (50-60%) of the total current but also caused a large slowing of the apparent activation rate that was reversed by a 50 ms prepulse to +160 mV (data not shown). These effects were not seen with treatment with lower concentrations of SNX-482 (<= 5 µM), and therefore data obtained by applying concentrations of SNX-482 >10 µM were not included in the concentration-response curve.



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Fig. 5. Effects of R-type calcium channel blocker SNX-482 on IBa in NT2-N cells. Whole cell currents evoked by 300-ms steps from a holding potential of -80 to +5 mV were studied for their sensitivity to SNX-482. A: current traces showing the reversible block of the control current (1) by various concentrations of SNX-482: 110 nM (2), 220 nM (3), and 1.1 µM (4). B: concentration-response curve for inhibition of calcium channel current by SNX-482. C: record of currents evoked by 300-ms steps from a holding potential of -80 to +5 mV from an 8-wk postdifferentiation NT2-N. The cell was then treated successively with omega -conotoxin GVIA (10 µM); omega -conotoxin MVIIC (10 µM); nifedipine (10 µM), and SNX-482 (1.1 µM). D: current traces of IBas evoked in C: (1) Control current; (2) omega -conotoxin GVIA (10 µM); (3) omega -conotoxin MVIIC (10 µM); (4) nifedipine (10 µM); (5) SNX-482 (1.1 µM).

SNX-482 (1.1 µM) substantially, but not completely, blocked the residual toxin-resistant IBa remaining after treatment with specific L-, N-, and P/Q-type blockers. Figure 5C shows a time course of current amplitude of an 8-wk-old NT2-N cell sequentially treated with toxins, and Fig. 5D shows current traces evoked from this cell. The maximal IBa of 472 pA (Fig. 5D, trace 1) was reduced to 402 pA by application of CTx GVIA (10 µM) from a puffer pipette (Fig. 5D, trace 2). The current did not recover when the pipette was removed from the recording medium. Subsequent applications of CTx MVIIC (10 µM) (Fig. 5D, trace 3) and nifedipine (10 µM, Fig. 5D, trace 4) resulted in a relatively inactivating current with an amplitude of 174 pA. Application of SNX-482 (1.1 µM, Fig. 5D, trace 5) substantially reduced this toxin-resistant current to an amplitude of 56 pA, (i.e., inhibition of ~70% of the "toxin-resistant" current) representing an inhibition of 24% of the original, pretoxin current.

To determine whether SNX-482 specifically inhibited R-type currents or also had effects on other calcium current subtypes, we compared the magnitude of SNX-482 inhibition of IBa before and after blockade of L-, N-, and P/Q-type channels. The effects of SNX-482 on "resistant" current are shown in Fig. 6A. Combined application of omega CTx GVIA (10 µM), omega CTx MVIIC (10 µM), and nifedipine (10 µM) inhibited ~62% of total IBa. This residual current was rapidly activating and rapidly inactivating (Fig. 6B), consistent with block of the slowly activating L-type channels. Subsequent treatment with SNX-482 (1.1 µM) substantially, but not completely, blocked the residual current. SNX-482 block accounted for ~29% of the original IBa and left a residual SNX-482-insensitive IBa of ~9% of the original current amplitude. Figure 6, C and D, shows the effects of SNX-482 on total IBa. The NT2-N cell described was treated with increasing concentrations of SNX-482 (22 nM, 220 nM, and 1.1 µM). SNX-482 maximally inhibited 36% of the original IBa. The cell was also treated with nifedipine (10 µM, 30 s) at the times marked (down-arrow ). Nifedipine inhibition was not substantially changed after SNX-482 treatment, indicating that SNX-482 does not block L-type channels. A mixture of CTx GVIA (10 µM) and MVIIC (10 µM) applied to block N- and P/Q-type channels inhibited a further 40% of IBa, and subsequent nifedipine (10 µM) further inhibited IBa (~19% of original IBa) to leave a small residual current (6% of original IBa). Figure 6E compares inhibition of IBa by 1.1 µM SNX-482 (calculated as percent of original, pretoxin IBa) before ("SNX first") and after ("CTx first") blockade of L-, N-, and P/Q-type currents. After blockade of N- and P/Q-type currents, there was a small but significant occlusion of maximal SNX-482 inhibition of IBa. This suggests that SNX-482 at a maximally inhibiting concentration (1.1 µM) has small effects on N- and P/Q-type channels; however, the majority of the SNX-482 inhibition appears to be due to effects on R-type channels.



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Fig. 6. Comparison of effects of SNX-482 on IBa in NT2-N cells before and after block of L-, N-, and P/Q-type channels. Whole cell IBa evoked by 300 ms steps from a holding potential of -80 to +5 mV were studied for their sensitivity to SNX-482 before and after block of N-, P/Q-, and L-type currents. A: record of peak IBa vs. time after patch rupture in an 8-wk NT2-N cell. The cell was treated with a mixture of GVIA (10 µM), MVIIC (10 µM), and nifedipine (10 µM), which inhibited ~60% of IBa. The residual current was further reduced by application of 1.1 µM SNX-482, representing ~29% of the peak pretoxin IBa. B: superimposed IBa traces evoked before toxin, after GVIA, MVIIC, and NIF, and after further application of SNX-482, as indicated. C: IBa vs. time in an 8-wk NT2-N cell. SNX-482 was applied in serial applications 22 nM, 220 nM, and 1.1 µM, as indicated (), resulting in maximal inhibition of 35% of IBa. Following SNX-482, the cell was treated with a mixture of GVIA (10 µM) and MVIIC (10 µM), which further reduced IBa by 32%. down-arrow , 30-s treatments with nifedipine (10 µM), which reversibly inhibited the equivalent of 25-30% of pretoxin IBa. D: superimposed IBa traces evoked before toxin and after treatment with SNX-482 (1.1 µM); GVIA (10 µM) + MVIIC (10 µM), and nifedipine (10 µM) as indicated. E: percent inhibition of total cellular IBa by 1.1 µM SNX-482, before (SNX first) or after (CTx first) blockade of N-, P/Q-, and L-type channels.

We also compared the effects of SNX-482 and nifedipine at Vh = -80, which produced maximal IBas, and at Vh = -50 mV, at which N and P/Q types of calcium channels previously have been shown to be substantially inactivated but at which L-type channels are only slightly inactivated (Fox et al. 1987). Concentration-response curves for nifedipine were obtained at Vh = -80 and -50 mV to determine the relative contribution of L-type currents at these holding potentials (Fig. 7A). Average percent inhibition of IBa was plotted as a function of nifedipine concentration and fit with a sigmoidal curve. Nifedipine blocked peak currents in a concentration-dependent manner at both holding potentials (Fig. 7A). Maximal inhibition by nifedipine at Vh = -50 mV (40.4 ± 2.5%, n = 5), was significantly greater (P < 0.05) than maximal inhibition at -80 mV (19 ± 1.2%, n = 30). Nifedipine (10 µM) did not completely block IBa evoked from a Vh = -50 mV, providing further evidence for the existence of a non-L, non-N-, non-P/Q-type current (i.e., R type) that was not completely inactivated at this holding potential. To further investigate the identity of the calcium channels subtypes activatible at Vh = -50 mV, we examined effects of SNX-482 at this holding potential. Figure 7B shows an NT2-N cell at Vh = -80 mV, at which a maximal IBa of 782 mV was evoked and then treated with nifedipine (10 µM, 30 s), which inhibited IBa by 168 pA or 21%. Changing the Vh to -50 mV reduced IBa to 279 pA. Application of SNX-482 (1.1 µM) inhibited 132 pA of this residual current, which represented 53% of the residual IBa, but only 17% of "total" IBa, i.e., IBa at Vh = -80 mV, indicating that R-type channels were partially inactivated at Vh = -50 mV. Application of 10 µM nifedipine reduced a further 102 pA or 13% of the "total" IBa. Thus while L-type currents were also somewhat inactivated at -50 mV, the IBa remaining at Vh = -50 mV was composed primarily of L- and R-type current, and N- and P/Q-type currents appeared to be almost completely inactivated. Figure 7, C-F, shows IBa traces from the same cell described in Fig. 7B. Figure 7C shows IBas evoked at Vh = -80 mV before and after nifedipine treatment. We isolated L-type current by subtraction of nifedipine-treated from control IBa at Vh = -80 mV (Fig. 7D). The current inactivating at Vh = -50 mV, i.e., presumably primarily N- and P/Q-type currents, was isolated by subtracting IBa at Vh = -50 mV from that at -80 mV (Fig. 7E). Figure 7F shows superimposed traces of IBa evoked at Vh = -50 mV before and after treatment with SNX-482 and nifedipine, as indicated.



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Fig. 7. SNX-482 and nifedipine block of residual IBa in NT2-N cells at Vh = -50 mV. A: concentration-response curves for nifedipine were generated on separate 1- to 2-wk-old NT2-N cells to compare the ability of nifedipine to block whole cell currents evoked by steps to +5 mV from Vh = -50 mV (, IC50 = 165 nM, n = 5 cells) vs. -80 mV (, IC50 = 56 nM, n = 4 cells). The curves represent logistic equations used to fit the data. B: effects of holding potential on IBa amplitude and sensitivity to inhibitors. Peak IBa is plotted vs. time. IBas were evoked from a 9-wk NT2-N cell held initially at Vh = -80 mV () and treated with nifedipine (10 µM, 30 s) as indicated. The holding potential was then changed to -50 mV (), which reduced maximal IBa amplitude. The cell was then treated with SNX-482 (1.1 µM) and nifedipine (10 µM). C: superimposed IBas evoked from the cell described in B at Vh = -80 mV before and after treatment with nifedipine. D: isolation of L-type current was obtained by subtracting the nifedipine-treated IBa from the untreated IBa. E: isolation of current inactivating at -50 mV was obtained by subtracting the IBa evoked from Vh = -50 mV from the IBa evoked from Vh = -80 mV. The resulting current is therefore the high-voltage-activated (HVA) calcium channel current that inactivated over this voltage range. The rapidly activating, rapidly inactivating current was 62% of the total IBa and was similar in time course to native N-type channel current. It presumably was composed of the majority of N-, P- and Q-type currents and a portion of R- and L-type currents. F: superimposed IBas evoked at Vh = -50 mV were untreated or treated with SNX-482 and nifedipine, as indicated.


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The NT2-N cell line is a terminally differentiated human cell line that exhibits many neuronal features, including neuronal morphology and the expression of multiple neuron-specific cytoplasmic and secretory markers (Pleasure and Lee 1993; Pleasure et al. 1992). NT2-N cells also express functional muscarinic, glutamate, and opioid receptors (Beczkowska et al. 1997; Wolf et al. 1995) as well as both ligand-gated (GABAA, kainate, and NMDA receptors) and voltage-gated (sodium channel) ion channels (Matsuoka et al. 1997; Neelands et al. 1998). Because NT2-N cells exhibit a neuron-like phenotype, are derived from an immortalized clonal cell line of human origin and can be transfected with exogenous recombinant genes (Cook et al. 1996), they may make an excellent model system for study of the regulation of ion channels in a human neuronal milieu.

NT2-N cells express HVA calcium currents

We characterized the calcium channel current subtypes expressed in NT2-N cells using whole cell electrophysiology. When cells were held at either -80 or -100 mV, no inward current was evoked until the cell membrane was depolarized above -35 mV. In addition, no slowly activating, rapidly inactivating currents were recorded, suggesting NT2-N cells did not express T-type currents.

Changes in the holding potential caused a nonlinear decrease in the maximal peak of the current-voltage relation similar to the steady-state inactivation curve (which was performed in a similar manner). This decrease was likely due to the differences in voltage-dependent inactivation among different calcium channel types. The remaining current had a progressive rightward shift in the voltage of half-activation. NT2-N cells, similar to acutely isolated and cultured neuronal preparations, exhibited several subtypes of HVA calcium currents that could be blocked by specific pharmacological agents. Calcium channel current amplitude increased with the age of the NT2-N cells after the 5-wk retinoic acid protocol, but the relative proportion of calcium current subtypes and the apparent activation and inactivation kinetics did not change substantially over time. The L-type channel blocker nifedipine inhibited a slightly higher portion of whole cell IBa in 8- to 10- versus 1- to 3-wk postdifferentiation (25 vs. 18%, respectively); in contrast, the partial inhibitions of IBa by the N-type channel blocker omega -conotoxin GVIA and the P/Q-type channel omega -conotoxin MVIIC were relatively constant (~30 and 15%, respectively). The nifedipine-sensitive current, obtained by subtracting the residual nifedipine-treated current trace from the untreated current trace, displayed little inactivation, consistent with L-type channel currents. Likewise, at Vh = -50 mV, the current was nearly completely blocked by nifedipine and SNX-482, suggesting that it was composed primarily of L- and R-type currents and that N- and P/Q-type channels were inactivated, as has previously been suggested (Fox et al. 1987). Thus the combined N-, P- and Q-type calcium currents could be isolated by subtracting the current traces evoked at Vh = -50 mV, at which these channel types are inactivated, from that at Vh = -80 mV. These current traces displayed the characteristic rapid activation and inactivation kinetics associated with these current types.

NT2-N cells express substantial R current

A substantial portion of the IBa (37 and 30% in 1- to 3- and 8- to 10-wk-old NT2-N, respectively) was not blocked by coapplication of nifedipine, omega -conotoxin GVIA, and omega -conotoxin MVIIC, consistent with the presence of toxin resistant current(s). Toxin-resistant currents have been pharmacologically isolated in a number of native neuronal preparations, including neurohypophyseal parvocellular neurons and cerebellar granule cells. The percentage of resistant current was similar to that reported for neurons of the nucleus accumbens (Churchill and MacVicar 1998) but was larger than the 19% reported in cerebellar granule neurons (Randall and Tsien 1997) or 7% reported in intracardiac neurons (Jeong and Wurster 1997). While all of the observed toxin-resistant currents have been referred to as R-type currents, recent evidence using SNX-482 demonstrates that R-type currents are not a homogenous current and may not be carried by a single channel species (Newcombe et al. 1998; Totenne et al. 2000). Tottene et al. (2000) report the presence of SNX-482 sensitive and insensitive "residual currents" in cerebellar neurons, consistent with our finding in NT2-N cells of components of residual current sensitive and resistant to 1.1 µM SNX-482.

Biophysical and pharmacological features of the toxin-resistant currents in NT2-N cells are consistent with previously described R-type currents. Recombinant alpha 1E calcium channels, believed to form R-type calcium channels, have been shown to have steady-state inactivation profiles similar to L-type channels (Qin et al. 1998). Assuming the relative proportions of total IBa of L- and R-type currents from Fig. 4C, that only L- and R-type currents were present at -50 mV and that the majority of these channels were not inactivated, then the R-type current would be predicted to be about 2/3 of the total current at Vh = -50 mV. This prediction is consistent with SNX-482 and nifedipine action when NT2-N cells were held at Vh = -50 mV. The SNX-482-sensitive current, as well as the current resistant to combined nifedipine and conotoxin treatment at -80 mV, showed considerable inactivation during the 200-ms step, as would be expected of currents from R-type channels.

The R-type channel blocker SNX-482 maximally inhibited 30-35% of the total whole cell current in NT2-N cells, consistent with SNX-482 inhibiting the same toxin-resistant calcium current component(s) insensitive to L-, N-, and P/Q-type blockers. Furthermore, SNX-482 inhibited ~75% of the residual current isolated by combined application of these toxins. These data strongly suggested the existence of R-type calcium current components. The majority of this current was sensitive to SNX-482 and thus possibly mediated by E-class calcium channels, but a non-SNX-482-sensitive component was also present, consistent with other reports of SNX-482 action (Newcombe et al. 1998; Tottene et al. 2000). The observed IC50 of total IBa in NT2-N by SNX-482 (119 nM) was substantially higher than the reported IC50 for IBa from cells transfected with the E-class alpha  subunit (30 nM) and the 90% maximal inhibition of toxin-resistant current from neurohypophyseal neurons (40 nM) (Newcomb et al. 1998) but is consistent with one component of SNX-482-sensitive current described in rat cerebellar granule cells (Tottene et al. 2000). A further caveat is that very high (>10 µM) concentrations of SNX-482 inhibited a higher proportion of the total IBa, possibly through effects on N-type current, and in contrast to inhibition by lower toxin concentrations, led to a substantial slowing of the apparent activation rate. Voltage prepulses to +85 mV caused a reversal of the kinetic slowing and a partial reversal of the magnitude of inhibition (data not shown), suggesting the presence of additional mechanism(s) of calcium channel inhibition at high concentrations of SNX-482.

Taken together these data suggest that an R-type current comprises a substantial proportion of the calcium current in NT2-N cells. The finding that SNX-482 did not completely inhibit the toxin resistant current in NT2-N cells is consistent with previous reports that SNX-482 completely inhibited current from cells expressing recombinant alpha 1E channels and L-, N- and P/Q-type channel blocker resistant current from neurohypophyseal cells but did not inhibit R-type currents from other cell types, including cerebellar granule cells (Newcomb et al. 1998). Our findings are also consistent with a recent report that cerebellar granule cells contain three distinct toxin-resistant components, two of which are sensitive to SNX-482 with differing affinities and a third that is resistant to SNX-482 (Tottene et al. 2000). These findings support the conclusion that there may be either multiple alpha 1 subunits that comprise the R-type current or that co-expression of auxiliary subunits can alter the pharmacological profile of the channel (Jones et al. 1998; Parent et al. 1997). The possibility of multiple alpha 1 subunits may explain the previous finding that NT2-N cells may lack alpha 1E subunit mRNA (Gao et al. 1998a,b), as only alpha 1D (L type) and alpha 1B (N type) subunit mRNAs were amplified by RT-PCR from NT2-N cells. Our data, however, indicate that NT2-N cells express an omega -conotoxin MVIIC-sensitive P/Q-type channel and suggest potentially two R-type (SNX-482 sensitive and insensitive) currents in addition to N- and L-type channels. The lack of signal from alpha 1E subunit in the previous study could have been due either to low levels of message for the P/Q/R-type channels as a result of low turnover of the protein or primer specificity. The alpha 1E subunit, for example, has both alternatively spliced carboxy termini (Williams et al. 1994) and different tissue specific isoforms (Vajna et al. 1998), so the primers utilized in previous studies may have not amplified the specific message(s) encoding the SNX-482-sensitive and insensitive R-type channels producing the currents recorded in this study.

Different calcium channels have been proposed to have specific cellular functions. For example, N-type calcium channels have been proposed to initiate synaptic vesicle fusion on activation by action potentials that depolarize the synaptic membrane. Therefore the differential expression of the various calcium channel types among different neuronal populations has been suggested to be important to the specific function of the cell. Little is known about the role of R-type calcium channels in neuronal functioning, but recent evidence suggests that they are important in dendritic calcium entry and synaptic transmission (Wu and Saggau 1994; Wu et al. 1998). Although the NT2-N cell line may not correspond directly to a specific type of native neuron, the high level of resistant calcium current in these cells may be helpful in elucidating the function of this current in a neuronal cell type.


    ACKNOWLEDGMENTS

We are grateful for the gifts of NT2 stem cells and NT2-N neurons and instructions on their care and use from Dr. R. Scott Turner and for indispensable assistance with cell culture from N. Esmaeil, J. E. Novak, A. Y. Yang, and J. Zhang. We also gratefully acknowledge the gift of SNX-482 from Dr. George Miljanic of Neurex, division of Elan Pharmaceuticals.

This work was supported by National Institute on Drug Abuse (NIDA) Grant R01-DA-0412211 to R. L. Macdonald. A.P.J. King is a recipient of NIDA Postdoctoral Fellowship 2T32DA-07268.

Present address of T. R. Neelands: University of Connecticut Health Center, Dept. of Pharmacology, MC-6125, 263 Farmington Ave., Farmington, CT 06030.


    FOOTNOTES

Address for reprint requests: R. L. Macdonald, Neuroscience Laboratory Bldg., 1103 E. Huron St., Ann Arbor, MI 48104-1687 (E-mail: rlmacd{at}umich.edu).

Received 18 October 1999; accepted in final form 31 August 2000.


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ABSTRACT
INTRODUCTION
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