1Neuroscience Program, 2Department of Neurology, and 3Department of Physiology, University of Michigan, Ann Arbor, Michigan 48104-1687
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-CTx-GVIA (Reynolds et al. 1986
), and N-,
P-, and Q-type currents are all reversibly inhibited by
-CTx-MVIIC
(Hillyard et al. 1992
). Preferential block by the spider
toxin
-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 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
1 subunit currents were similar to those of native calcium channel type currents
(L-type:
1C,
1D;
N-type:
1B; Q/P-type:
1A; R-type:
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
2(A-E)/
,
1-4, and
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 1B (N type) and
1D (L type) mRNA and cytosolic calcium
transients that were blocked by nifedipine and
-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.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M 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
-conotoxin-GVIA (
-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.
-CTx-GVIA and
-conotoxin-MVIIC (
-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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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
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
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
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.
|
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).
|
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
-CTx GVIA, nifedipine,
-CTx MVIIC, and/or cadmium
applied with pressure ejection (
-CTx GVIA), weeper pipettes (
-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
-CTx GVIA (10 µM) was applied first and then removed from the recording media to spare P/Q currents.
-CTx MVIIC (10 µM), a toxin that blocks N-, P-, and Q-type calcium channels, was applied following treatment by
-CTx GVIA, thus making
it specific for P/Q-type channels. In the 1-wk postdifferentiation NT2-N cell analyzed in Fig.
4A,
-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
-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.
|
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).
-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).
-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
-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.
|
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
CTx GVIA (10 µM),
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 (
). 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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-conotoxin GVIA and the P/Q-type channel
-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, -conotoxin GVIA, and
-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 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
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
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
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
1 subunits may explain the previous
finding that NT2-N cells may lack
1E subunit
mRNA (Gao et al. 1998a
,b
), as only
1D (L type) and
1B (N
type) subunit mRNAs were amplified by RT-PCR from NT2-N cells. Our
data, however, indicate that NT2-N cells express an
-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
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
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|