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Volume 16, Number 20,
Issue of October 15, 1996
pp. 6353-6363
Copyright ©1996 Society for Neuroscience
Functional Diversity of P-Type and R-Type Calcium Channels in Rat
Cerebellar Neurons
Angelita Tottene,
Alessandra Moretti, and
Daniela Pietrobon
Department of Biomedical Sciences and Consiglio Nazionale delle
Ricerche Center of Biomembranes, University of Padova, 35131 Padova,
Italy
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
By combining single-channel and whole-cell patch-clamp recordings,
we have established the sensitivity to  -agatoxin IVA and
-conotoxin MVIIC (SNX-230) of G1, G2, and G3, the three novel
non-L-, non-N-type Ca2+ channels characterized previously
in rat cerebellar granule cells. G1 channels were blocked irreversibly
by both -conotoxin MVIIC and low doses of -agatoxin IVA
(saturation at 50 nM). Thus, according to pharmacological
criteria, G1 channels must be classified as P-type Ca2+
channels. Being slowly inactivating during depolarizing pulses and
completely inactivated at voltages in which steady-state inactivation
of P-type channels in Purkinje cells is negligible, G1 represents a
novel P subtype. Neither G2 nor G3 was blocked irreversibly by
-conotoxin MVIIC, and therefore both are R-type Ca2+
channels. G2 and G3 have some biophysical properties similar to those
of low-voltage-activated (LVA) Ca2+ channels (e.g., voltage
range for steady-state inactivation,
V1/2 =  90 mV), some properties similar to
those of high-voltage-activated (HVA) Ca2+ channels (e.g.,
high sensitivity to Cd2+ block), and other properties
intermediate between those of LVA and HVA Ca2+ channels,
with LVA properties prevailing in G2 and HVA properties prevailing in
G3. The R-type whole-cell current was inhibited by Ni2+
with a biphasic dose-response curve (IC50: 4 and 153 µM), suggesting that G2 and G3 may have a different
sensitivity to Ni2+ block. Our results uncover functional
diversity of both native P-type and R-type Ca2+ channels
and show that R subtypes with distinct biophysical properties are
coexpressed in rat cerebellar granule cells.
Key words:
calcium channel;
cerebellum;
granule neuron;
patch clamp;
conotoxin;
agatoxin;
channel diversity
INTRODUCTION
The fundamental roles played by voltage-gated
Ca2+ channels in neuronal physiology and pathology (Llinas,
1988 ; Augustine and Neher, 1992; Johnston et al., 1992; Lipton and
Rosenberg, 1994; Dunlap et al., 1995; Ghosh and Greenberg, 1995)
explain the large and prolonged effort spent to study the functional,
pharmacological, and molecular diversity of brain Ca2+
channels.
According to pharmacological criteria, neuronal high-voltage-activated
(HVA) Ca2+ channels have been classified as dihydropyridine
(DHP)-sensitive channels (L-type), -conotoxin GVIA (GVIA)-sensitive
channels (N-type), and -agatoxin IVA (-AgaIVA)-sensitive channels
(P- and Q-type, distinguished on the basis of different sensitivities
to -AgaIVA; Kd < 10 nM for
P-type and Kd > 10 nM for Q-type)
(Birnbaumer et al., 1994; Olivera et al., 1994; Dunlap et al., 1995;
Randall and Tsien, 1995). An additional component of HVA current
(R-type) has been identified as the current resistant to DHPs and
-conotoxin MVIIC (SNX-230, Neurex Corporation, Menlo Park, CA)
(MVIIC; Hillyard et al., 1992; Zhang et al., 1993).
Cloning studies have shown that brain Ca2+ channels
 1 and  subunits are encoded by at least five
(1A, 1B, 1C,
1D, 1E) and four (1,
2, 3, 4) different genes,
respectively (Birnbaumer et al., 1994). Further molecular diversity is
created by the existence of multiple splice variants for each gene
(Perez-Reyes and Schneider, 1994). Heterologous expression studies have
shown that 1C and 1D give rise to
DHP-sensitive Ca2+ channels, 1B to
GVIA-sensitive Ca2+ channels, 1A to
-AgaIVA-sensitive Ca2+ channels, and 1E
to Ca2+ channels resistant to all specific inhibitors
(Mikami et al., 1989; Williams et al., 1992a,b; Sather et al., 1993;
Soong et al., 1993; Perez-Reyes and Schneider, 1994). Moreover, they
have shown that combinations of a given 1 subunit with
different subunits give rise to Ca2+ channels with
different biophysical properties (Sather et al., 1993; Olcese et al.,
1994; Stea et al., 1994; De Waard and Campbell, 1995).
Considering the different isoforms for 1 and subunits and the
many possible subunit combinations, a functional diversity of brain
Ca2+ channels much wider than the established
pharmacological diversity can be expected in native neuronal membranes.
Although there is evidence for functional heterogeneity of neuronal
L-type (Forti and Pietrobon, 1993; Kavalali and Plummer, 1994), N-type
(Nowycky et al., 1985; Plummer et al., 1989; Carbone et al., 1990;
Kasai and Neher, 1992; Elmslie et al., 1994), and T-type
Ca2+ channels (Carbone and Lux, 1984; Huguenard and Prince,
1992; Huguenard et al., 1993), functional diversity of P-type channels
has not been reported yet. Actually, the lack of inactivation in long
depolarizations and the steady-state inactivation at relatively
positive voltages of P-type channels in Purkinje cells (Regan, 1991;
Usowicz et al., 1992) are considered characteristic properties of
P-type channels, distinguishing them from Q-type channels. On the other
hand, faster inactivation together with lower sensitivity to -AgaIVA
have been considered distinctive features of Q-type channels with
respect to P-type channels (Sather et al., 1993; Randall and Tsien,
1995).
Recently, we have characterized the single-channel properties of three
novel DHP- and GVIA-insensitive Ca2+ channels coexpressed
in rat cerebellar granule cells (Forti et al., 1994). We have called
these channels G1, G2, G3 (in which G stands for granule and 1, 2, 3 refer to the order of frequency with which they were observed), because
their biophysical properties were different from those of both P- and
T-type Ca2+ channels. G1, G2, and G3 showed some
inactivation during long depolarizations and complete steady-state
inactivation at voltages in which steady-state inactivation of P-type
channels is negligible. We have now assessed the sensitivity of G1,
G2, and G3 to -AgaIVA and MVIIC. Our results demonstrate that,
according to pharmacological criteria, G2 and G3 are R-type
Ca2+ channels, whereas G1 channels are P-type
Ca2+ channels and represent a novel P subtype with
biophysical properties distinct from the subtype expressed in Purkinje
cells.
MATERIALS AND METHODS
Cell culture. Cerebellar granule cells were grown in
primary culture after enzymatic and mechanical dissociation from 6- to
7-d-old Wistar rats according to the procedure of Levi et al. (1984).
The cells were plated on poly-L-lysine-coated glass
coverslips and kept in basal Eagle's medium supplemented with 10%
fetal calf serum, 25 mM KCl, 2 mM glutamine,
and 60 µg/ml gentamycin. Cytosine arabinoside (10 µM)
was added to the culture 18 hr after plating to inhibit the
proliferation of non-neuronal cells. Granule cells were the large
majority of the cells in the cultures and were morphologically
identified by their oval or round cell body, small size, and bipolar
neurites. Experiments were performed on granule cells grown from 5 to
8 d in vitro (DIV), with the majority of experiments at
6-7 DIV.
Patch-clamp recordings and data analysis. Single-channel and
whole-cell patch-clamp recordings followed standard techniques (Hamill
et al., 1981). Currents were recorded with a DAGAN 3900 patch-clamp
amplifier, low pass-filtered at 1 kHz (3 dB; 8-pole Bessel filter),
sampled at 5 kHz, and stored for later analysis on a PDP-11/73
computer. Experiments were performed at room temperature
(21-25°C).
All single-channel recordings were obtained in the cell-attached
configuration. Linear leak and capacitative currents were subtracted
digitally from all records used for analysis. Current amplitude
histograms were obtained from the data directly, with bin width equal
to our maximal resolution (323.6 points/pA). For display, each
histogram was normalized to the value of the zero current peak. Open
probability, Po, was computed by measuring the
average current  I in a given single-channel current
record and dividing it by the unitary single-channel current
i. To obtain activation curves, Po
values were calculated by averaging the open probabilities measured in
each sweep at a given voltage only in segments with single-channel
activity. In this case the values of Po reflect
the voltage-dependent equilibrium between short-lived open and closed
states in the activation pathway. Open-channel current amplitudes for
i-V were measured by manually fitting cursors to
well-resolved channel openings. A channel opening or closure was
detected when more than one sampling point crossed a discriminator line
at 50% of the elementary current. Histograms of open and closed times
were fit with sums of decaying exponentials, as described in Forti and
Pietrobon (1993).
The pipette solution contained (in mM): 90 BaCl2, 10 TEACl, 15 CsCl, and 10 HEPES, pH 7.4 (with
TEAOH). The bath solution contained (in mM): 140 K-gluconate, 5 EGTA, 35 L-glucose, and 10 HEPES, pH 7.4 (with KOH). The high-potassium bath solution was used to zero the
membrane potential outside the patch. In some experiments the bath
solution contained 0.5-1 µM (+)-(S)-202-791.
Liquid junction potential at the pipette tip was +12 mV (pipette
positive), and this value should be subtracted from all voltages to
obtain the correct values of membrane potentials in cell-attached
recordings (Neher, 1992). For assessing the sensitivity of single
channels to MVIIC, the toxin was usually added to both the pipette
solution and the divalent-free bath solution. Cells were always
incubated for at least 10 min in the presence of toxin before
recording.
For whole-cell recordings, cells were placed into a recording chamber
with Tyrode's solution and, after attainment of the whole-cell
configuration, were perfused with the external recording solution
containing (in mM): 5 BaCl2, 147.5 TEA-Cl,
and 10 HEPES (adjusted to pH 7.4 with TEA-OH). Internal solution
contained (in mM): 100 Cs-methanesulfonate, 5 MgCl2, 30 HEPES, 10 EGTA, 4 ATP, 0.5 GTP, and 1 c-AMP
(adjusted to pH 7.4 with CsOH). The perfusion system consisted of six
microcapillary Teflon tubes glued together and placed inside a standard
plastic pipette (Gilson, Middleton, WI) at ~12 mm from the tip
(~1.2 mm diameter), which was cut to have a flute-beak shape and
positioned close to the cell. The tubes were fed by gravity from
reservoirs containing external solution with or without toxins.
Switching between different solutions was controlled by solenoid
valves. Delay time for complete solution change was <8 sec. Cytochrome
C (0.1 mg/ml) was included in all recording solutions to block
nonspecific peptide-binding sites. Barium currents were corrected for
leak and capacitative currents by subtraction of an appropriately
scaled current elicited by a 10 mV hyperpolarization. Averages are
given as mean ±SEM. Liquid junction potential at the pipette tip was
8 mV (pipette negative) and that between the Tyrode's solution in
the experimental chamber and the external recording solution (flowing
from the capillary tube) was 4 mV; these two junction potentials
should be added to all voltages to obtain the correct values of
membrane potential in whole-cell recordings (Neher, 1992). Isolated
cells were chosen for recording. We considered space clamp as adequate
when notch-like current discontinuities were absent from recordings at
different voltages and when slow components in the decay of
capacitative currents (in response to a hyperpolarizing pulse) or in
the decay of tail currents (in the presence of nimodipine; Forti and
Pietrobon, 1993) either were absent or had a negligible amplitude. The
experiment was discarded if these criteria were not met.
All drugs were stored as stock solutions at 20°C: 3 mM
(+)-(S)-202-791 (gift from Dr. Hof, Sandoz, Basel,
Switzerland) in 95% ethanol, 250 µM -conotoxin GVIA
(Peninsula Laboratories, Belmont, CA, and Bachem, Budendorf,
Switzerland) in distilled water, 250 µM -conotoxin
MVIIC (SNX-230 provided by Neurex Corporation) in distilled water, 95 µM -AgaIVA (gift from M. Adams, University of
California at Riverside, Riverside, CA in first single-channel
experiments, and then a gift from Pfizer, Groton, CT, in majority of
whole-cell experiments; some experiments were done with the toxin sold
by Bachem, Budendorf, Switzerland) in distilled water, and 10 mM nimodipine (gift from Dr. B. Bean, Vollum Institute,
Portland, OR) in ethanol absolute.
RESULTS
Table 1 summarizes the single-channel properties of
G1, G2, and G3, the three novel DHP- and GVIA-insensitive
Ca2+ channels we have recently characterized in rat
cerebellar granule cells in primary culture (Forti et al., 1994).
Besides differing in unitary current, conductance, and mean open time,
G1, G2, and G3 channels also differ in activation threshold, which
increases in the order G2 (40 mV with 90 mM
Ba2+ as charge carrier) < G3 (25 mV) < G1 (10 mV).
Steady-state inactivation of G1 channels occurs at relatively negative
voltages (over the range from 80 to 30 mV) and that of G2 and G3
channels at even more negative voltages, with complete inactivation
already at holding potentials of 50 mV. G channels inactivate rather
slowly: the percentage of decay of average single-channel current at
the end of 720-msec-long depolarizations to +10 mV does not reach 50%
for the most rapidly inactivating G2 channels.
To complete the pharmacological characterization of G1, G2, and G3
channels and to classify them as either P/Q or R-type Ca2+
channels, we assessed their sensitivity to MVIIC, a toxin that inhibits
irreversibly both P- and Q-type channels. Establishing the individual
sensitivity of G1, G2, and G3 channels to this membrane-impermeable
conopeptide is not straightforward, because G-type channels run down in
excised patches, and, as an additional problem, binding of MVIIC to
P/Q-type channels is inhibited by high divalent ion concentrations.
Using human neuroblastoma IMR32 cells, which are rich in N-type
channels (Carbone et al., 1990), we have established previously an
experimental protocol effective in blocking single N-type
Ca2+ channels in cell-attached patches (Forti et al.,
1994). Before they were recorded, the cells were incubated in the
divalent-free bath recording solution in the presence of 2.3 µM GVIA for at least 10 min. Irreversible block of N-type
channels was inferred from the absence of these channels in 70 cell-attached patches from cells preincubated with toxin, compared with
their presence in 17 of 69 patches from control cells (Forti et al.,
1994). We concluded that, once bound, the rate of unbinding of the
toxin was sufficiently slow to allow detection of channel block in
cell-attached recordings. Given the established irreversibility of
block of P/Q-type channels by MVIIC (compare Fig. 3), we used here the
same protocol. After incubation for at least 10 min with 10 µM MVIIC in the potassium gluconate bath recording
solution, G1 channels were never observed in 51 cell-attached patches
(with 3 µM MVIIC in the pipette). In paired controls
without toxin performed alternatively on the same neuronal preparation
at each day in culture, they were observed in 16 of 40 patches
(excluding all multichannel patches in which identification was
uncertain). We can then conclude that G1 Ca2+ channels are
blocked irreversibly by MVIIC. In contrast, because G2 and G3
Ca2+ channels were still observed after incubation with
MVIIC and their activity was similar to that in control patches (Fig.
1), neither G2 nor G3 is blocked irreversibly by MVIIC.
According to the accepted pharmacological classification of HVA
Ca2+ channels into four classes (L, N, P/Q, R), each of
which may comprise channel subtypes with distinct biophysical
properties, both G2 and G3 are R-type Ca2+ channels.
Fig. 3.
Pharmacological properties of DHP-insensitive
whole-cell Ba2+ current of rat cerebellar granule cells.
Whole-cell recordings with 5 mM Ba2+ as charge
carrier in the continuous presence of 3-5 µM nimodipine
to inhibit L-type channels. a, Plot of peak
Ba2+ current versus time for an experiment in which 1 µM GVIA and 3 µM MVIIC were sequentially
applied and then washed; examples of traces from the same experiment
were taken at times indicated by a, b, c.
Inset, Calibration: 50 pA, 20 msec. Depolarizations 56 msec long (Vt = 10 mV) were delivered
every 10 sec from Vh = 90 mV. Cell T84D.
Shown on the right are representative traces recorded
during 720-msec-long depolarizations (from a different experiment: cell
U22H; calibration: 50 pA, 80 msec). Trace a, Total
DHP-insensitive current, 38% current decay after 720 msec,
biexponential best fit with  1 = 50 msec,
2 = 1938 msec, and relative amplitudes of 0.5 and 99.5%
(average values: 1 = 46 ± 3 msec, 2 = 1979 ± 51 msec; relative amplitudes 0.4 and 99.6%;
n = 11). Trace c, Resistant current
in the presence of MVIIC and GVIA, 57% decay after 720 msec,
biexponential best fit with 1 = 40 msec,
2 = 1177 msec, and relative amplitudes of 1.3 and 98.7%
(average values: 1 = 66 ± 11 msec,
2 = 1196 ± 104 msec; relative amplitudes 2 and
98 ± 1%, respectively; n = 4). Traces
a-c, Difference current, 31% decay after 720 msec,
biexponential best fit with 1 = 57 msec,
2 = 2000 msec, and relative amplitudes 0.3 and 99.7%.
b, Dose-response curve for Ni2+ inhibition
of the resistant current recorded in the presence of (in
µM): 5 nimodipine, 1 GVIA, and 5 MVIIC. Data were pooled
from five cells. The data points were best fit by the sum of two
Langmuir adsorption isotherms with dissociation constants
Kd1 = 11.8 µM and
Kd2 = 286 µM, number of
binding sites n1 = 1.9 and
n2 = 1.1, and relative amplitudes 52 and
48%, respectively. Representative traces of resistant current during
720-msec-long depolarizations (Vt = 10 mV;
Vh = 90 mV) before and after addition of
100 µM Ni2+ are shown on the
bottom (calibration: 50 pA, 80 msec). Percentage of
decay (67%) and time course of decay (1 = 78 msec,
2 = 1029 msec) of the resistant current inhibited by 100 µM Ni2+ were similar to those of the total
resistant current. Cell U13C.
[View Larger Version of this Image (15K GIF file)]
Fig. 1.
G2 and G3 Ca2+ channels of rat
cerebellar granule cells are not blocked irreversibly by MVIIC.
Cell-attached recordings with 90 mM Ba2+ as
charge carrier. Shown are representative current traces and normalized
current amplitude histograms from all traces with activity of single G2
and G3 channels at 10 mV. Depolarizations were 720 msec long and were
delivered every 4 sec from holding potentials of 80 to 100 mV.
Records were sampled and filtered at 5 and 1 kHz, respectively.
Control: G2, unitary current i = 0.69 pA, average open probability Po = 0.20, cell N03C; G3, i = 1.05 pA,
Po = 0.06, cell N56B [in the presence of 1 µM (+)-(S)-202-791 in the incubation
bath]. MVIIC: Cell-attached recordings with 3 µM MVIIC in pipette after incubation of neurons with 10 µM MVIIC in the potassium gluconate bath solution [and
in the presence of 0.5 µM
(+)-(S)-202-791]. G2, i = 0.68 pA,
Po = 0.23, one L-type channel was also
present in the patch (compare unresolved openings of higher amplitude
in second trace), cell U24A; G3, i = 0.95 pA,
Po = 0.06, cell U23D. In paired control and
MVIIC recordings (i.e., performed alternatively on the same neuronal
preparation at each day in culture), G2 channels were observed in 9 of
42 patches in controls and in 4 of 32 patches with MVIIC; G3 channels
were observed in 8 of 40 patches in controls and in 4 of 31 patches
with MVIIC (excluding all multichannel patches for which identification
was uncertain). G2 and G3 channels were identified on the basis of
their unitary current and conductance and on the basis of the voltage
range for activation (compare Table 1 and Forti et al.,
1994).
[View Larger Version of this Image (18K GIF file)]
Recently, Randall and Tsien (1995) subdivided the MVIIC-sensitive
current of cerebellar granule cells into two components on the basis of
their different sensitivities to -AgaIVA: a noninactivating P-type
component inhibited by -AgaIVA with a Kd ~1
nM (as in Purkinje cells) and a slowly inactivating Q-type
component inhibited by -AgaIVA with a Kd
~90 nM. The biophysical properties of our MVIIC-sensitive
G1 channels are closer to those of the Q-type component as defined by
Randall and Tsien than to those of P-type channels in Purkinje cells.
However, because the pharmacological distinction between P- and Q-type
channels is based on different affinities for -AgaIVA, it was
necessary to assess the sensitivity of G1 channels to -AgaIVA before
classifying them as either Q-type channels or P-type channels
functionally different from those of Purkinje cells.
The interpretation of results obtained by using single-channel
recordings and a protocol similar to that used with MVIIC to
investigate the sensitivity of G1 channels to -AgaIVA is complicated
by the relatively small difference in affinity for -AgaIVA of P- and
Q-type channels, which was established on the basis of whole-cell
recordings with low Ba2+ concentrations and the difficulty
in predicting the relative affinities of P- and Q-type channels in the
presence of high Ba2+ concentrations. An additional
complicating factor is the voltage dependence of -AgaIVA binding
(Mintz et al., 1992b), which makes it difficult to predict the relative
affinities of P- and Q-type channels in the depolarizing K-gluconate
bath solution used in our cell-attached recordings. Moreover, in our
hands, the presence of -AgaIVA in the pipette solution led to
destabilization of the seal. We therefore investigated the sensitivity
of G1 channels to -AgaIVA with whole-cell recordings after
ascertaining that both functional and pharmacological properties of the
DHP-insensitive whole-cell Ba2+ current were similar to
those of G channels (G1 + G2 + G3) in cell-attached patches.
Figure 2 shows the voltage-dependent properties of the
whole-cell current recorded with 5 mM Ba2+ as
charge carrier in the continuous presence of 3 µM
nimodipine. At this concentration, nimodipine inhibited on average
24 ± 2% (n = 12) of total current (at holding
potential Vh = 90 mV and test potential
Vt = 10 mV) and was saturating for L-type
current inhibition (data not shown; cf. Amico et al., 1995). Given the
small fraction of DHP-insensitive current blocked irreversibly by GVIA
(10 ± 2%, n = 10), this current should be
attributable mainly to Ba2+ influx through G1, G2, and G3,
the three types of channels observed most frequently (together with
L-type) in cell-attached patches of cerebellar granule cells (Forti and
Pietrobon, 1993; Forti et al., 1994). In reasonable agreement with the
steady-state activation and inactivation properties of single G-type
channels, significant DHP-insensitive inward current was recorded by
depolarizing the membrane above 50 mV (from holding potential
Vh = 90 mV; Fig. 2b), and
this current was reduced progressively to almost zero by increasing the
holding potential from 110 to 40 mV (Fig.
2c).
Fig. 2.
Biophysical properties of DHP-insensitive
whole-cell Ba2+ current of rat cerebellar granule cells.
Whole-cell recordings with 5 mM Ba2+ as charge
carrier in the continuous presence of 3-5 µM nimodipine
to inhibit L-type channels. a, Representative traces at
increasing test depolarizations (Vt = 50
to 10 mV) from holding potential Vh = 90
mV (top) and representative traces at
Vt = 10 mV from different holding
potentials (Vh = 40 to 100 mV) in the
same cell (cell U04A). Depolarizations were 56 msec long and were
delivered every 10 sec. The traces at different
Vh were recorded after the attainment of the
new steady-state value after each change of
Vh (i.e., after at least 1 min from the
change). b, Peak normalized Ba2+ current
In as a function of
Vt (Vh = 90
mV). Data were pooled from five cells. For each cell the current was
normalized with respect to maximal peak current at 10 mV. The
dashed lines are the fractional contributions to
whole-cell current of G1, G2, and G3 at different
Vt, predicted from their single-channel
properties, assuming that the DHP-insensitive current was entirely
attributable to G channels. The contribution of each channel was
calculated as i × Po × % obs × (100 % nulls) at each
Vt, in which i is the unitary
current, Po is the open probability in
active traces, % obs is the frequency of observation in cell-attached
patches, and % nulls is the fraction of traces without activity at
Vh = 90 mV [values taken from Forti et
al. (1994); compare Table 1] and assuming an average of 20 mV more
negative surface potential at the lower Ba2+ concentration
of whole-cell with respect to single-channel recordings.
c, Peak normalized Ba2+ current
In as a function of holding potential
Vh (Vt = 10
mV). Data were pooled from five cells. For each cell the current was
normalized with respect to maximal current at
Vh = 110 mV. The data points were best fit
by a Boltzmann distribution function of the form
In = In max × {1 + exp [(V V1/2)/k]} 1
with V1/2 = 74.9 mV, k = 16.9 mV, and In max = 1.12 (continuous line). The dashed line is the
sum of two Boltzmann functions (see below). d, Peak
Ba2+ current as a function of Vt
at Vh = 90 mV (circles) and
Vh = 60 mV (squares). Cell
T43E. e, Steady-state inactivation curves at
Vt = 30 mV (squares), at
Vt = 10 mV (circles), and
Vt = +10 mV (triangles). Cell
U21B. Peak currents were normalized with respect to the current at
Vh = 100 mV for each
Vt. The steady-state inactivation curve at
30 mV was best fit by a Boltzmann function with
V1/2 = 85.5 mV, k = 11.5 mV, and In max = 1.3. The steady-state
inactivation curves at 10 and +10 mV were fit by Boltzmann functions
with V1/2 = 73.6, k = 15.3, In max = 1.18; and
V1/2 = 63.7, k = 13.7, In max = 1.07 (continuous
lines). Dashed lines are best fits with two
Boltzmann distributions. The best fit of the steady-state inactivation
curve at +10 mV was obtained with
(V1/2)2 = 55.9 mV and
k2 = 10.8, using for
(V1/2)1 and
k1 the values obtained from the best fit of
the steady-state inactivation curve at 30 mV (relative amplitudes
from best fit: 33 and 67% for components 1 and 2, respectively).
Although two Boltzmann functions did not fit better than one, the
steady-state inactivation curve at 10 mV, using
(V1/2)1 = 85.5 mV,
k1 = 11.5 mV and
(V1/2)2 = 55.9 mV,
k2 = 10.8, the best fit was obtained with
relative amplitudes of 58 and 42% for components 1 and 2, respectively. Using as parameters for the two Boltzmann functions the
average values obtained from three experiments
[(V1/2)1 = 90.4,
k1 = 10.9;
(V1/2)2 = 58.2,
k2 = 11.8], we obtained the best fit of the
average inactivation curve at 10 mV (dashed line in
c) with relative amplitudes of 46 and 54% for
components 1 and 2, respectively.
[View Larger Version of this Image (25K GIF file)]
From the combined more negative voltage range for steady-state
inactivation and more negative voltage threshold for activation of G2
and G3 channels with respect to G1, one expects that (1) the threshold
for current activation should shift toward more positive voltages if
the holding potential is changed toward less negative values, and (2)
the voltage range of steady-state inactivation should shift toward more
positive voltages if the test depolarization is changed from values
(e.g., 30 mV) in which almost all of the current is predicted to be
through G2 and G3 channels to values (e.g., +10 mV) in which most of
the current is predicted to be through G1 channels (see Fig.
2b, fractional contributions to whole-cell current of
G1, G2, and G3 predicted from their single-channel properties and their
relative abundance in cell-attached patches). These predictions were
confirmed by our results. When the holding potential was changed from
90 to 60 mV, significant DHP-insensitive inward current started to
be recorded at more positive voltages (Fig. 2d).
Moreover, the fractional current inactivation at
Vh = 60 mV was reduced progressively from 80 to 45% with increasing Vt in the range from
30 to 10 mV (the range in which the fractional contribution to
whole-cell current of G2 plus G3 channels is expected to decrease and
that of G1 to increase). When the test depolarization was changed from
30 to +10 mV, the voltage range of steady-state inactivation was
shifted toward more positive voltages (Fig. 2e). An
intermediate steady-state inactivation curve was obtained at
Vt = 10 mV, in which G1 and G2 plus G3
channels are expected to contribute similar amounts of whole-cell
current. In agreement with the inactivation properties of G2 and G3
channels, steady-state inactivation of the current at 30 mV occurred
over a very negative voltage range (V1/2 = 90.4 ± 1.7, n = 3) and was almost complete at
Vh = 60 mV. Fitting steady-state inactivation
curves at +10 mV with two Boltzmann distributions provided a value of
V1/2 = 58.2 ± 1.7 (n = 3) for the Boltzmann distribution at more positive voltages, which
should describe steady-state inactivation of G1 channels in isolation
(with some negligible contribution from the small fraction of N-type
channels). In good agreement with the prediction on the basis of
single-channel properties of G channels, steady-state inactivation
curves at 10 mV were best fit (using the parameters obtained for G2
plus G3 and G1 currents in isolation), assuming a similar contribution
of G2 plus G3 and G1 channels to the whole-cell current at 10 mV (see
legend, Fig. 2e).
Besides the biophysical properties, the pharmacological properties of
the DHP-insensitive whole-cell current of cerebellar granule cells are
also consistent with the conclusion that most of this current is
attributable to Ba2+ influx through G1, G2, and G3 channels
(Fig. 3). GVIA inhibited only a relatively small
component of DHP-insensitive current (average total inhibition 20 ± 2%, n = 25; Vh = 90 mV;
Vt = 10 mV) and, in general, only a fraction
of it in an irreversible manner (average irreversible inhibition
10 ± 2%, n = 10). In contrast, as expected from
the irreversible block by MVIIC of G1 channels, MVIIC added after GVIA
inhibited slowly and irreversibly a large component of DHP-insensitive
whole-cell current (43 ± 2%, n = 20). The
average time constant of inhibition by 3 µM MVIIC, added
after GVIA, was 87 ± 11 sec (n = 6). As expected
from single-channel recordings, a large component of current, similar
in amplitude to that inhibited by MVIIC, was resistant to both GVIA and
MVIIC (38 ± 2%, n = 21). In agreement with the
inactivation kinetics of single G2 channels, this resistant whole-cell
current inactivated relatively slowly (on average 56 ± 4% at the
end of 720-msec-long depolarizations to 10 mV, n = 4)
and displayed a biexponential time course. As expected from the
relatively slower inactivation of G1 channels with respect to G2 (Table
1), the percentage of decay of the total DHP-insensitive current was
smaller than that of the resistant current (38 ± 1%,
n = 11), and the time constant of the primarily
prevailing slow component of the biexponential inactivation time course
was larger (see legend, Fig. 3a).
Figure 3b shows that the resistant current consisted of at
least two components with differential sensitivity to Ni2+
block, one extremely sensitive and inhibited by Ni2+ with
an IC50 of 3.8 µM, the other less sensitive
and inhibited by Ni2+ with an IC50 of 153 µM. The two components contributed approximately equally
to the resistant current.
Because the biophysical and pharmacological properties of the
DHP-insensitive whole-cell current and of single G channels are quite
similar, we can assess whether G1 channels belong to the
pharmacological category of P- or Q-type by studying the effect of low
doses of -AgaIVA on whole-cell Ba2+ current. We used 30 nM -AgaIVA, a concentration that should be saturating
for block of P-type channels and only partially inhibitory for Q-type
channels, and based our discrimination between P- and Q-type channels
on the absence or presence of further inhibition of whole-cell current
by higher doses of -AgaIVA.
Figure 4 shows that -AgaIVA concentrations above 30 nM hardly caused further inhibition of DHP-insensitive
whole-cell current. Inhibition by -AgaIVA was not removed by wash
(Fig. 4b) but could be completely removed by two series of
ten 50-msec-long depolarizations to +130 mV (indicated by P
in Fig. 4a). The average time constant of inhibition by 30 nM -AgaIVA was 80 ± 12 sec (n = 5). The properties of -AgaIVA block, including time course,
saturation at 50 nM, and voltage dependence, are quite
similar to those reported previously in cerebellar Purkinje cells and
used to define P-type Ca2+ channels (Mintz et al.,
1992a,b). We decided to distinguish between P- and Q-type pharmacology
on the basis of the absence or presence of further inhibition by
concentrations of -AgaIVA higher than 30 nM and not on
the basis of the effect of lower concentrations of toxin, because it
was difficult to separate the very slow kinetics of inhibition by  10
nM -AgaIVA from those (variable from cell to cell) of
rundown and to establish when steady-state inhibition had been reached.
However, we measured the inhibition of the DHP-insensitive current
produced by sequential additions of 5 and 50 nM -AgaVA
in six neurons with negligible rundown. After 5 min from the addition
(a time that was sufficient to reach steady-state inhibition with 50 nM, but not with 5 nM toxin), 50 nM
-AgaIVA inhibited 52 ± 3% of non-L current (in good agreement
with the data in Fig. 4c from a different group of 12 cells), and 5 nM -AgaIVA inhibited 16 ± 1% of the
same current. Thus, 5 nM -AgaIVA inhibits at least
31 ± 2% of the total -AgaIVA-sensitive component of
whole-cell current of cerebellar granule cells, in good agreement with
the data reported by Mintz et al. (1992a) in cerebellar Purkinje cells
(see their Fig. 3c).
Fig. 4.
Inhibition by -AgaIVA of whole-cell
Ba2+ current of rat cerebellar granule cells.
a, Plot of peak Ba2+ current versus time for
an experiment in which 30, 50, and 100 nM -AgaIVA were
applied sequentially and then washed. After washing, two series of ten
50-msec-long depolarizations to +130 mV separated by 3 msec at
Vh = 90 mV and 10 msec at
Vt = 10 mV were applied (indicated by
P). Nimodipine (3 µM) was present
throughout. Examples of traces from the same experiment, taken at times
indicated by a, b, c, d, are shown on the
right (calibration: 100 pA, 20 msec). Cell T56E.
b, Plot of peak Ba2+ current versus time for
an experiment in which 100 and 200 nM -AgaIVA were
applied sequentially and then washed. Vh = 100 mV; Vt = 10 mV. Cell C66A.
c, Average inhibition of DHP-insensitive current by 30 nM -AgaIVA (44 ± 3%, n = 15),
50 nM -AgaIVA (54 ± 3%, n = 12), and 100 nM -AgaIVA (52 ± 3%,
n = 11). The averages at different concentrations
of toxin were obtained from the same preparations. Statistical
significance of 10% difference of inhibition between 30 and 50 nM -AgaIVA: p < 0.03; Student's
t test.
[View Larger Version of this Image (22K GIF file)]
If, as suggested by the data in Figure 4, Q-type Ca2+
channels are not present in significant amount in our cerebellar
granule cells, then (1) the average fraction of current inhibited by 50 nM -AgaIVA should be quantitatively similar to that
inhibited by 3 µM MVIIC added after GVIA, and (2) low
doses of -AgaIVA should occlude most of the slow and irreversible
block produced by MVIIC. Figure 5 shows that both
predictions were verified by our data. We conclude that MVIIC and low
doses of -AgaIVA inhibit the same channels and that these
Ca2+ channels can be classified pharmacologically as P-type
channels.
Fig. 5.
MVIIC and low doses of -AgaIVA inhibit the same
type of Ca2+ channels in rat cerebellar granule cells.
Whole-cell recordings with 5 mM Ba2+ as charge
carrier in the continuous presence of 3-5 µM nimodipine.
Depolarizations 56 or 136 msec long were delivered every 10 sec.
Vt = 10 mV; Vh = 90 mV. a, Filled bars, Average
fractional inhibition of DHP-insensitive current by 1 µM
GVIA (reversible + irreversible; 20 ± 2%, n = 25), by 50 nM -AgaIVA (47 ± 3%,
n = 20), by 3 µM MVIIC added after
GVIA (MVIIC*; 43 ± 2%, n = 20), and average fractional current remaining in the presence of
nimodipine plus GVIA plus MVIIC (R; 38 ± 2%,
n = 21). Empty bars, Sum of
pharmacological current components indicated below. The sum of the
average fraction of DHP-insensitive current resistant to GVIA and MVIIC
and the average fraction of current inhibited by GVIA and that
inhibited by low doses of -AgaIVA amounted to 104 ± 7%. In
agreement with Amico et al. (1995) and in contrast with Pearson et al.
(1995), there seems to be no significant overlapping selectivity
between GVIA and -AgaIVA. b, Plot of peak
Ba2+ current versus time for an experiment in which 50 nM -AgaIVA plus 2 µM GVIA and 3 µM MVIIC were sequentially applied and then washed.
Vh = 90 mV; Vt = 10 mV. Inset, Traces taken at times a, b,
c (calibration: 50 pA, 20 msec). Cell T82F. On average, the
fraction of non-L current inhibited irreversibly by 3 µM
MVIIC added after 50 nM -AgaIVA (+GVIA) was 10 ± 1% (n = 5).
[View Larger Version of this Image (24K GIF file)]
In agreement with the almost complete inactivation of whole-cell
current at Vh = 40 mV (Fig.
2c), our single-channel data show a relatively high
density of G1 channels inhibited irreversibly by MVIIC and no evidence
for the presence of noninactivating P-type channels [because the rare
noninactivating single channels mentioned in Forti et al. (1994) were
not blocked by MVIIC; our unpublished results]. We then expect that
the channels inhibited by MVIIC and low doses of -AgaIVA are G1
channels. Indeed, the biophysical properties of the whole-cell current
component inhibited by 30 nM -AgaIVA are similar to
those of G1 channels and quite different from those of P-type channels
in Purkinje cells, both in inactivation kinetics and in steady-state
inactivation properties (Fig. 6; compare also Fig. 3).
Taking advantage of the complete removal of -AgaIVA block by a
series of positive prepulses, we could measure in the same cell the
amount of current inhibited by -AgaIVA (30 nM) at two
different holding potentials (90 and 60 mV). Figure 6 shows that
the amount of current inhibited while holding at 90 mV (126 pA) was
approximately twice that inhibited while holding at 60 mV (66 pA), as
expected for G1 channels, a fraction of which should be inactivated at
60 mV and contrary to what is expected for P-type channels in
Purkinje cells that do not display steady-state inactivation in the
range 90 to 60 mV (Regan, 1991; Usowicz et al., 1992). On average,
the amount of current inhibited at 90 mV by low doses of -AgaIVA
was 1.9-fold ± 0.03 (n = 3) that inhibited at
60 mV. We can, therefore, conclude that G1 channels are P channel
subtypes with functional properties different from those of Purkinje
cells.
Fig. 6.
The Ca2+ channels inhibited by low
doses of -AgaIVA have inactivation properties similar to those of G1
channels. a, Plot of peak Ba2+ current
versus time for an experiment in which the holding potential was varied
as indicated, and the inhibition by 30 nM -AgaIVA was
measured first at Vh = 60 mV and then at
Vh = 90 mV, after removal of previous
block by a series of ten 50-msec-long depolarizations to +130 mV
(indicated by P). Nimodipine (3 µM) was
present throughout. Vt = 10 mV. Cell T48C.
b, Representative traces taken at times indicated
by a, b in a and corresponding
difference current. Calibration: 50 pA, 20 msec.
[View Larger Version of this Image (21K GIF file)]
Figure 7 displays the properties of a small component of
current, measurable only in some cells, which was inhibited rapidly and
reversibly by low concentrations of MVIIC. Panel a shows
that 100 nM MVIIC inhibited reversibly both the component
of current inhibited reversibly by 1 µM GVIA and a small
additional component, which amounted on average to 10 ± 1%
(n = 9) of the total DHP-insensitive current. Panel
b shows that the rapid and reversible block by MVIIC of this
component was not occluded by 50 nM -AgaIVA (+GVIA).
Given the rapidity (complete block in <1 min with 100 nM
MVIIC) and reversibility of block, this small component does not have
the properties of a Q-type current as defined in Randall and Tsien
(1995) and is similar to the ``Q-like'' current described by Magnelli
et al. (1995).
Fig. 7.
Properties of a small current component inhibited
rapidly and reversibly by MVIIC. Plots of peak Ba2+ current
versus time from whole-cell recordings in the continuous presence of 3 µM nimodipine. Depolarizations 56 msec long were
delivered every 10 sec. Vt = 10 mV;
Vh = 90 mV. a, GVIA (1 µM) inhibited 17% of non-L current. Of this inhibition,
50% was recovered by wash and was reinhibited by a subsequent addition
of the same concentration of GVIA. As shown by the fraction of current
recovered after wash, 100 nM MVIIC inhibited reversibly
both the component of current inhibited reversibly by GVIA and an
additional 11% of total non-L current. Inset, Traces
taken at times a, b, c, d (calibration: 50 pA, 20 msec).
Cell T93F. b, The reversible inhibition by MVIIC is not
occluded by low doses of -AgaIVA. In the presence of 50 nM -AgaIVA and 1 µM GVIA, MVIIC (3 µM) inhibited rapidly and reversibly 15% of total non-L
current. Inset, Traces taken at times a, b, c,
d. Calibration: 100 pA, 20 msec. Cell T91E.
[View Larger Version of this Image (17K GIF file)]
The pharmacological properties of G1, G2, and G3 channels, as derived
from this and our previous study (Forti et al., 1994), are summarized
in Table 2.
DISCUSSION
By combining single-channel and whole-cell patch-clamp recordings,
we have established the sensitivity to -AgaIVA and MVIIC of G1, G2,
and G3, the three novel non-L, non-N-type Ca2+ channels
recently characterized in rat cerebellar granule cells in primary
culture (Forti et al., 1994; Table 1). We have shown that neither G2
nor G3 channels are blocked irreversibly by MVIIC, whereas G1 channels
are blocked irreversibly by both MVIIC and low doses of -AgaIVA.
The characteristics of -AgaIVA block of G1 channels, namely its
saturation at 50 nM toxin, its relatively fast kinetics,
its irreversibility by prolonged wash, and its relief by prepulses to
very positive voltages, are all similar to those reported for
-AgaIVA block of P-type channels in Purkinje cells and other neurons
(Mintz et al., 1992a,b; Umemiya and Berger, 1994). Thus, according to
pharmacological criteria, G1 channels must be classified as P-type
rather than Q-type Ca2+ channels, although they are slowly
inactivating during depolarizing pulses and are completely inactivated
at voltages in which steady-state inactivation of P-type channels in
Purkinje cells is negligible (Regan, 1991; Usowicz et al., 1992). Our
data do not exclude a small difference in affinity for -AgaIVA
between G1 channels and P-type channels of Purkinje cells, but they do
exclude that such difference would exceed a factor of 10 and thus be
larger than the differences in affinities reported for different N or L
channel subtypes (Welling et al., 1993; Boland et al., 1994; Dunlap et
al., 1995).
In contrast to Randall and Tsien (1995), our data do not support the
subdivision of the -AgaIVA-sensitive whole-cell current of
cerebellar granule cells into a noninactivating P-type component
inhibited by -AgaIVA with a Kd ~1
nM and a slowly inactivating Q-type component inhibited
with a Kd ~90 nM (cf. also Pearson
et al., 1995). The reason for the conflicting result remains uncertain.
It might be attributable simply to expression of different
Ca2+ channels in cerebellar granule cells maintained in
culture under different conditions (in particular, different
concentration of K+ ions in culture medium).
Alternatively, because the actual data of Randall and Tsien (1995) show
at most only 10-15% additional inhibition by 1 µM
-AgaIVA with respect to 100 nM, most of the conflict
might be only apparent and be partly resolved if one admits that (1)
the assumption that -AgaIVA inhibits with high affinity only a
noninactivating current may not be right (as shown by our data), and
the consequent fitting of doses-responses with two one-to-one binding
curves may lead to an overestimation of the Kd
for the inactivating component, and (2) block of this component is
saturated at 100 nM -AgaIVA, and the additional small
fraction of current inhibited at higher concentrations of toxin may be
attributable to block of other Ca2+ channels, perhaps the
same channels that account for the small component inhibited rapidly
and reversibly by MVIIC shown in Figure 7. These channels do not fit
into any previously described pharmacological class of Ca2+
channels (except maybe the O-type of Olivera et al., 1994; cf. Magnelli
et al., 1995).
By showing that G1 channels represent a novel P channel subtype, our
results uncover functional diversity of native P-type channels and
should warn against considering lack of inactivation and steady-state
inactivation at relatively positive voltages as distinctive properties
of P-type channels on the one hand and slow inactivation and
steady-state inactivation at relatively negative voltages as
distinctive properties of Q-type channels on the other hand. The
recognition of P channel subtypes with distinct biophysical properties
is important, because they are expected to be differentially activated
during different patterns of neuronal activity.
Given the good correlation between densities of -AgaIVA-sensitive
currents and levels of 1A mRNA and 1A
immunostaining in rat cerebellar Purkinje and granule cells (Stea et
al., 1994; Westenbroek et al., 1995), it is very likely that both the
noninactivating P subtype of Purkinje cells and the slowly inactivating
P subtype (G1) of granule cells are encoded by 1A. It
remains unknown whether the different biophysical properties of the two
native P subtypes are exclusively attributable to different subunits (Sather et al., 1993; Stea et al., 1994; Yatani et al., 1994;
De Waard and Campbell, 1995) or whether different 1A
splice variants contribute to the functional diversity. Interestingly,
a high density of 1A has been found in the terminals of
the parallel fibers of rat cerebellar granule cells (Westenbroek et
al., 1995), and Ca2+ channels with a P-type pharmacology
have been shown to mediate 50% of Ca2+ influx controlling
neurotransmitter release at the granule cell to Purkinje cell synapse
in rat cerebellar slices (Mintz et al., 1995). Whether the P channel
located in the terminals is the same inactivating subtype (G1) that we
have characterized in the bodies of granule cells is unknown.
Besides functional diversity of P-type Ca2+ channels, our
study uncovers functional diversity of R-type Ca2+
channels, because both G2 and G3 are resistant to DHPs, GVIA, and
MVIIC. G2 and G3 differ mainly in single-channel conductance and
unitary current and in voltage range for activation (see Table 1).
Given the biphasic dose-response curve for Ni2+ block of
the whole-cell current resistant to nimodipine, GVIA, and MVIIC (Fig.
3), they might differ also in Ni2+ sensitivity, suggesting
that a relatively high Ni2+ sensitivity may not be a
general property of all R subtypes. In contrast with the fast
inactivation reported for R-type current in cerebellar granule cells by
Zhang et al. (1993), both G2 and G3 inactivate relatively slowly (Fig.
3; Forti et al., 1994). However, in a minority of granule cells, we
have observed a small component of whole-cell current inactivating
rapidly as in Zhang et al. (1993; data not shown), suggesting further
heterogeneity of R-type channels. Functional diversity of native R-type
channels is consistent with the widely different biophysical properties
reported for 1E coexpressed with different subunits
(Soong et al., 1993; Olcese et al., 1994; Wakamori et al., 1994).
Neither a classification as LVA Ca2+ channels nor as HVA
Ca2+ channels seems appropriate for the two R subtypes of
cerebellar granule cells (especially for G2). The quite negative
voltage range for steady-state inactivation of G2 channels
(V1/2 = 90 mV) is rather similar to that
reported for T-type currents in different CNS neurons (Akaike et al.,
1989; Mogul and Fox, 1991; Regan, 1991; Huguenard and Prince, 1992). On
the other hand, the slow inactivation and the high-affinity block by
Cd2+ of both G2 and G3 (Forti et al., 1994) associate them
to HVA Ca2+ channels rather than to LVA Ca2+
channels, which have in common a much faster inactivation (complete in
50-300 msec) and a smaller sensitivity to Cd2+ block
(Bossu et al., 1985; Fox et al., 1987; Huguenard and Prince, 1992).
Unitary currents and conductance and especially voltage range for
activation of G2 and G3 are intermediate between those of LVA and HVA
channels, with values closer to those of LVA for G2 and to those of HVA
for G3 (Table 1).
Given the low threshold for activation, G2 channels are expected to be
activated by subthreshold EPSPs, as recently shown for LVA channels in
dendrites of hippocampal and cortical neurons (Markram and Sakmann,
1994; Magee and Johnston, 1995). Moreover, given their steady-state
inactivation properties, their contribution to EPSP- or action
potential-evoked Ca2+ transients is expected to be strongly
dependent on previous hyperpolarization or prolonged depolarization of
the neuronal membrane. Thus, G2 channels may have a potentially
important role in the generation of calcium spikes, in synaptic
integration, and in postsynaptic forms of plasticity known to involve
Ca2+ influx.
The ranges for voltage-dependent activation and inactivation of G1, G2,
and G3 vary gradually and form almost a continuum along the voltage
axis. Thus it is not surprising that both current-voltage and
steady-state inactivation curves of DHP-insensitive Ca2+
current of granule cells (Fig. 2b,c) do not show
signs of multiple kinetic components. However, the presence of multiple
Ca2+ channels with slightly different voltage-dependent
properties becomes apparent from the different current-voltage
relations obtained at different holding potentials and the different
steady-state inactivation curves obtained at different test
depolarizations (Fig. 2d,e). Because the fractional
contribution of P and R Ca2+ channel subtypes to the
whole-cell current are expected to be quite different at different test
depolarizations and holding potentials (compare Fig. 2b),
some of the differences in functional and pharmacological properties of
whole-cell Ca2+ current reported for cerebellar granule
cells in different laboratories may be attributable to the different
voltage protocols adopted (De Waard et al., 1991; Slesinger and
Lansman, 1991; Rossi et al., 1994; Amico et al., 1995; Pearson et al.,
1995; Randall and Tsien, 1995).
The results reported in this and our previous articles (Forti and
Pietrobon, 1993; Forti et al., 1994) show that each of the
pharmacological classes of native neuronal Ca2+ channels
comprises various members with distinct biophysical properties and that
Ca2+ channels with similar pharmacological but distinct
biophysical properties can be coexpressed in a single type of neuron.
It seems likely that the large potential for combinatorial structural
heterogeneity of brain Ca2+ channels revealed by molecular
biology may indeed be fully exploited in native neuronal membranes.
Structural and functional heterogeneity of neuronal Ca2+
channels provides significant flexibility in the fine tuning of
Ca2+-dependent functions and may be the basis for selective
modification of local Ca2+-dependent events during specific
patterns of neuronal activity.
FOOTNOTES
Received May 6, 1996; revised July 9, 1996; accepted July 24, 1996.
The financial support of Telethon-Italy (Grants 392 and 720) to D.P. is
gratefully acknowledged. This work was also partially supported by
grants from the Regione del Veneto (Giunta Regionale-Ricerca Sanitaria
Finalizzata-Venezia-Italia) and the Italian Research Council (Consiglio
Nazionale delle Ricerche) Target Project Aging to D.P.; it is in
partial fulfillment of the doctoral programs of the University of
Padova in Molecular and Cellular Biology and Pathology (A.T.) and in
Pharmacology and Toxicology (A.M.). We thank Drs. J. R. Bell and L. Nadasdi of Neurex Corporation for providing -conotoxin MVIIC
(SNX-230), Dr. N. A. Saccomano of Pfizer for providing the -AgaIVA
used in most of our experiments, and Dr. M. Adams for providing the
-AgaIVA used in our initial experiments. We thank Drs. E. Carbone,
G. Carmignoto, and B. Hivert for critically reading this
manuscript.
Correspondence should be addressed to Dr. Daniela Pietrobon, Department
of Biomedical Sciences, University of Padova, Via Trieste 75, 35131 Padova, Italy.
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September 1, 1999;
19(17):
7268 - 7277.
[Abstract]
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R. L. Spaetgens and G. W. Zamponi
Multiple Structural Domains Contribute to Voltage-dependent Inactivation of Rat Brain alpha 1E Calcium Channels
J. Biol. Chem.,
August 6, 1999;
274(32):
22428 - 22436.
[Abstract]
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H. Grabsch, A. Pereverzev, M. Weiergräber, M. Schramm, M. Henry, R. Vajna, R. E. Beattie, S. G. Volsen, U. Klöckner, J. Hescheler, et al.
Immunohistochemical Detection of {alpha}1E Voltage-gated Ca2+ Channel Isoforms in Cerebellum, INS-1 Cells, and Neuroendocrine Cells of the Digestive System
J. Histochem. Cytochem.,
August 1, 1999;
47(8):
981 - 994.
[Abstract]
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J. Magistretti, S. Brevi, and M. de Curtis
Biophysical and pharmacological diversity of high-voltage-activated calcium currents in layer II neurones of guinea-pig piriform cortex
J. Physiol.,
August 1, 1999;
518(3):
705 - 720.
[Abstract]
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S. C. Molitor and P. B. Manis
Voltage-Gated Ca2+ Conductances in Acutely Isolated Guinea Pig Dorsal Cochlear Nucleus Neurons
J Neurophysiol,
March 1, 1999;
81(3):
985 - 998.
[Abstract]
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M. Hans, S. Luvisetto, M. E. Williams, M. Spagnolo, A. Urrutia, A. Tottene, P. F. Brust, E. C. Johnson, M. M. Harpold, K. A. Stauderman, et al.
Functional Consequences of Mutations in the Human alpha 1A Calcium Channel Subunit Linked to Familial Hemiplegic Migraine
J. Neurosci.,
March 1, 1999;
19(5):
1610 - 1619.
[Abstract]
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T G J Allen
The role of N-, Q- and R-type Ca2+ channels in feedback inhibition of ACh release from rat basal forebrain neurones
J. Physiol.,
February 15, 1999;
515(1):
93 - 107.
[Abstract]
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L.-G. Wu, R. E. Westenbroek, J. G. G. Borst, W. A. Catterall, and B. Sakmann
Calcium Channel Types with Distinct Presynaptic Localization Couple Differentially to Transmitter Release in Single Calyx-Type Synapses
J. Neurosci.,
January 15, 1999;
19(2):
726 - 736.
[Abstract]
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D. R Ireland, P. J Davies, and E. M McLachlan
Calcium channel subtypes differ at two types of cholinergic synapse in lumbar sympathetic neurones of guinea-pigs
J. Physiol.,
January 1, 1999;
514(1):
59 - 69.
[Abstract]
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Y. M. Nakashima, S. M. Todorovic, D. F. Covey, and C. J. Lingle
The Anesthetic Steroid (+)-3alpha -Hydroxy-5alpha -androstane-17beta -carbonitrile Blocks N-, Q-, and R-Type, but Not L- and P-Type, High Voltage-Activated Ca2+ Current in Hippocampal and Dorsal Root Ganglion Neurons of the Rat
Mol. Pharmacol.,
September 1, 1998;
54(3):
559 - 568.
[Abstract]
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E. S. Piedras-Renteria and R. W. Tsien
Antisense oligonucleotides against alpha 1E reduce R-type calcium currents in cerebellar granule cells
PNAS,
June 23, 1998;
95(13):
7760 - 7765.
[Abstract]
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L.-G. Wu, J. G. G. Borst, and B. Sakmann
R-type Ca2+ currents evoke transmitter release at a rat central synapse
PNAS,
April 14, 1998;
95(8):
4720 - 4725.
[Abstract]
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H. Moreno, B. Rudy, and R. Llinas
beta subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line
PNAS,
December 9, 1997;
94(25):
14042 - 14047.
[Abstract]
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E. D'Angelo, G. De Filippi, P. Rossi, and V. Taglietti
Synaptic Activation of Ca2+ Action Potentials in Immature Rat Cerebellar Granule Cells In Situ
J Neurophysiol,
September 1, 1997;
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[Abstract]
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L. R. Shekter, R. Taussig, S. E. Gillard, and R. J. Miller
Regulation of Human Neuronal Calcium Channels by G Protein beta gamma Subunits Expressed in Human Embryonic Kidney 293 Cells
Mol. Pharmacol.,
August 1, 1997;
52(2):
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[Abstract]
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