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The Journal of Neuroscience, July 15, 1998, 18(14):5240-5252
G-Protein-Dependent Facilitation of Neuronal  1A,
1B, and 1E Ca Channels
Ulises
Meza and
Brett
Adams
Department of Physiology and Biophysics, University of Iowa College
of Medicine, Iowa City, Iowa 52242-1109
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ABSTRACT |
Modulation of neuronal voltage-gated Ca channels has important
implications for synaptic function. To investigate the mechanisms of Ca
channel modulation, we compared the G-protein-dependent facilitation of
three neuronal Ca channels.  1A,
1B, or 1E subunits were
transiently coexpressed with 2- b and
 3 subunits in HEK293 cells, and whole-cell currents were
recorded. After intracellular dialysis with GTP S, strongly
depolarized conditioning pulses facilitated currents mediated by each
Ca channel type. The magnitude of facilitation depended on current
density, with low-density currents being most strongly facilitated and
high-density currents often lacking facilitation. Facilitating
depolarizations speeded channel activation ~1.7-fold for
1A and 1B and increased current amplitudes by the same proportion, demonstrating equivalent
facilitation of G-protein-inhibited 1A and
1B channels. Inactivation typically obscured
facilitation of 1E current amplitudes, but the
activation kinetics of 1E currents showed consistent and
pronounced G-protein-dependent facilitation. The onset and decay of
facilitation had the same kinetics for 1A,
1B, and 1E, suggesting that
G dimers dissociate from and reassociate with these Ca channels
at very similar rates. To investigate the structural basis for N-type
Ca channel modulation, we expressed a mutant of 1B
missing large segments of the II-III loop and C terminus. This
deletion mutant exhibited undiminished G-protein-dependent
facilitation, demonstrating that a G interaction site recently
identified within the C terminus of 1E is not required for modulation of 1B.
Key words:
Ca channel modulation; neuronal Ca channels; membrane-delimited pathway; G-protein-dependent Ca channel inhibition; presynaptic inhibition; signal transduction; neuronal integration; neuronal plasticity; molecular neuroscience; facilitation; 1A; 1B; 1C; 1E; neurosecretion; electrical excitability
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INTRODUCTION |
Voltage-gated Ca channels play
essential roles in neurosecretion and other neuronal functions (Dunlap
et al., 1995 ). At least six different classes of Ca channel
1 subunits (1A,
1B, 1C, 1D, 1E, and
1G) are expressed in neurons, in which they
contribute to the formation of native P/Q-, N-, L-, R-, and T-type Ca
channels, respectively (Hofmann et al., 1994; Perez-Reyes et al.,
1998). The activities of N-type and P/Q-type channels are known to be modulated by G-protein-dependent pathways (Elmslie et al., 1990; Sah,
1990; Bernheim et al., 1991; Mintz and Bean, 1993), and such modulation
is likely to have considerable physiological importance (cf. Kavalali
et al., 1997; Koh and Hille, 1997; Wu and Saggau, 1997).
Previous studies in neurons have identified five G-protein-dependent
pathways for N-type Ca channel inhibition (Hille, 1994). One pathway is
membrane-delimited and may involve only Ca channels, heterotrimeric
G-proteins, and neurotransmitter-hormone receptors. Ca channels
inhibited via this pathway exhibit positive shifts in the voltage
dependence of activation, slowed activation kinetics, and reduced
macroscopic current amplitudes; such channels are described as being
"reluctant" to open (Bean, 1989). Reluctant channels can be
transiently reconverted into "willing" channels by strong or
sustained depolarization (Bean, 1989; Elmslie et al., 1990; Ikeda,
1991); this reconversion is known as facilitation.
G-protein-dependent modulation has been extensively studied for native
N-type Ca channels (cf. Jones and Elmslie, 1997), and modulation of
cloned 1A and 1B Ca channels has been
reconstituted in expression systems (Zhou et al., 1995; Zong et al.,
1995; Patil et al., 1996; Brody et al., 1997; Herlitze et al., 1997;
Page et al., 1997). Interestingly, when neurotransmitter receptors are
used to activate G-proteins in a phasic manner, 1B
channels are more strongly inhibited and more strongly facilitated than 1A channels (Zhang et al., 1996; Zamponi et al., 1997).
To further examine the relative sensitivities of 1A and
1B to G-protein-mediated inhibition, we have compared
modulation of these channels by G-proteins tonically activated with
GTPS. Under these conditions, 1A and 1B display very similar magnitudes and kinetics of
facilitation, suggesting that other factors in addition to channel
primary structure may influence Ca channel-G-protein interactions.
Membrane-delimited Ca channel modulation appears to be effected by
G rather than G subunits (Herlitze et al., 1996; Ikeda, 1996;
Shekter et al., 1997). It has been proposed that direct interaction
with G occurs at the cytoplasmic I-II loop (De Waard et al.,
1997; Zamponi et al., 1997), the C terminus (Qin et al., 1997), or a
combination of the first transmembrane domain and the C terminus of the
Ca channel 1 subunit (Zhang et al., 1996; Page et al.,
1997). To investigate this issue, we have further studied facilitation
of a deletion mutant of 1B. This N-type Ca channel,
which lacks large segments of the II-III loop and C terminus, exhibits
undiminished G-protein-dependent facilitation, demonstrating that a
G interaction site recently identified within the C terminus of
1E (Qin et al., 1997) is not essential for modulation of
1B.
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MATERIALS AND METHODS |
Cell culture and transfection. Human embryonic kidney
(HEK293) cells (CRL 1573) were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and maintained at 37°C in a
humidified atmosphere containing 5% CO2. The culture
medium contained 90% DMEM (Life Technologies, Gaithersburg, MD;
catalog #11995-065), 10% heat-inactivated horse serum (Life
Technologies; catalog #26050-13), and 50 µg/ml gentamicin (Life
Technologies; catalog #15710-015). Every 2-3 d, the cells were briefly
trypsinized and replated at fourfold lower density. At the time of
replating, 35 mm culture dishes (Falcon; catalog #3002) were seeded
with ~103 cells per dish. Approximately 16 hr
later, these cells were transfected using the Ca-PO4
precipitation technique (Pharmacia, Piscataway, NJ; Cell Phect Kit)
with expression plasmids encoding 1A (rabbit brain; Mori
et al., 1991), 1B (rabbit brain; Fujita et al., 1993), 1C (rabbit heart; Mikami et al., 1989), or
1E (BII-2, rabbit brain; Niidome et al., 1992) at 1 µg
of each cDNA per dish. Cells were simultaneously cotransfected with
expression plasmids encoding 2-b (rat
brain; Kim et al., 1992) and 3 (rabbit brain; Witcher et
al., 1993) at 1 µg of each cDNA per dish and also with plasmid EBO-pCD-Leu2 encoding human CD8 protein (ATCC; catalog #59565) at 0.2 µg/dish. Cells expressing CD8 were visually identified by their
ability to bind 4.5 µm diameter paramagnetic beads coated with
anti-CD8 antibody (Dynal, Great Neck, NY). Decorated cells were
selected for electrophysiological analysis (Jurman et al., 1994).
Expression plasmids. The amino acid compositions and
construction of expression plasmids encoding 1A,
1B, and 1C have been described
previously (Tanabe et al., 1990; Fujita et al., 1993; Adams et al.,
1994). cDNAs encoding these 1 subunits were in the
expression vector pKCRH2 (Mishina et al., 1984). The entire coding
sequence of 1E was excised from pSPCBII-2 (Wakamori et al., 1994) using HindIII and EcoRI; the resulting
~7.3 kb fragment was ligated into the corresponding sites of
pcDNA3.1+ (Invitrogen, San Diego, CA). The
construction of pKCRBIII-DD, encoding a double-deletion mutant of
1B (1B-DD), has been previously described (Zhou et al., 1995). 1B-DD is missing amino
acid residues 829-995 and 1877-2338 from the II-III loop and C
terminus, respectively. The cDNA encoding
2-b (Kim et al., 1992) was in pMT2. The
cDNA encoding 3 was in pcDNA3.
Electrophysiology. Large-bore pipettes were pulled from 100 µl borosilicate micropipettes (VWR Scientific; catalog #53432-921) and filled with a solution containing (in mM:) 155 CsCl, 10 Cs2 EGTA, 4 Mg ATP, and 10 HEPES, pH 7.4, with CsOH. The
pipette solution also contained Li-GTPS (0.32 mM) or
Li-GDPS (0.30 mM) as noted. Aliquots of pipette
solutions were stored at  80°C and kept on ice after thawing.
Pipette solutions were filtered at 0.22 µm immediately before use.
Pipette tips were coated with paraffin to reduce capacitance and then
fire-polished; filled pipettes had DC resistances of 1.0-1.5 M . The
bath solution contained (in mM:) 145 NaCl, 40 CaCl2, and 10 HEPES, pH 7.4, with NaOH. Residual
pipette capacitance was compensated in the cell-attached configuration
using the negative capacitance circuit of the Axopatch 200A amplifier.
No corrections were made for liquid junction potentials. Temperature
(20-23°C) was continuously monitored using a miniature thermocouple
placed in the bath.
Ca currents were recorded using the whole-cell patch-clamp technique
(Hamill et al., 1981). The steady holding potential was normally 90
mV. In all experiments involving GTPS, cells were dialyzed for  5
min before studying G-protein-dependent effects. Currents were filtered
at 2-10 kHz using the built-in Bessel filter (four-pole low-pass) of
the Axopatch 200A amplifier and sampled at 10-50 kHz using a Digidata
1200 analog-to-digital board installed in a Gateway 486-66V computer.
The pCLAMP software programs Clampex and Clampfit (version 6.0.3) were
used for data acquisition and analysis, respectively. Figures were made
using Origin (version 4.1).
Linear cell capacitance (C) was determined by
integrating the area under the whole-cell capacity transient, evoked by
clamping from 90 to 80 mV with the whole-cell capacitance
compensation circuit of the Axopatch 200A turned off. The average value
of C was 22 ± 1 pF (n = 155 cells).
Series resistance (RS) was calculated as
(1/C) ×  , where is the time constant for decay of
the whole-cell capacity transient. Cells exhibiting more than one were rejected. Because pipette resistances and cell capacitances were
relatively small, was usually <100 µsec, and
RS was <5 M without using the series
resistance compensation circuit of the amplifier; when required, this
circuit was used to reduce and RS by
30-80%. The average values of and RS in
the reported experiments (n = 155) were 71 ± 4 µsec and 3.3 ± 0.1 M, respectively. Because maximal Ca
currents were typically <1 nA, voltage errors were usually <5 mV. The
DC resistance of the whole-cell configuration was routinely >1 G.
All illustrated and analyzed currents have been corrected for linear
capacitance and leakage currents using the P/6 method. Current
densities (expressed in picoamperes per picofarad) were calculated as
peak Ca current divided by C. Time constants for activation
of Ca currents were estimated by fitting the activating phase of
currents with a single exponential function.
A standard "facilitation" voltage protocol was used, consisting of
two identical test pulses (P1 and P2) separated by a conditioning pulse
(CP) to +100 mV (Fig. 1). Unless
otherwise noted P1, P2, and CP were each 25 msec long and separated by
10 msec repolarizations to 90 mV. This voltage protocol induced
maximal facilitation (see Fig. 9). Successive episodes of the voltage
protocol were separated by 10 sec intervals.
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Figure 1.
Representative whole-cell Ca currents recorded
from HEK293 cells expressing 1A or 1B
channels, illustrating G-protein-dependent facilitation. Currents were
recorded after 5 min of intracellular dialysis with GTPS or
GDPS, as indicated. The voltage protocol is diagrammed at top
left. P1, P2, and
CP were each 25 msec in duration and were separated by
10 msec intervals. A, 1A with GTPS,
data file 97425003; C = 20 pF;
RS = 2.7 M. 1A with
GDPS, data file 97D24008; C = 18 pF;
RS = 3.2 M. B,
1B with GTPS, data file 97403026;
C = 16 pF; RS = 4.8 M. 1B with GDPS, data file 97D19038;
C = 23 pF; RS = 2.0 M.
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Statistical analysis. Groups of data were compared using
one-way ANOVA or a two-tailed, unpaired t test, as
appropriate. Averaged data are presented in the text and figures as
mean ± SEM.
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RESULTS |
Facilitation of 1A and 1B
Figure 1 illustrates whole-cell Ca currents mediated by
1A or 1B Ca channels coexpressed with
2-b and 3 subunits in HEK 293 cells. After dialyzing cells with GTPS for several minutes, 1A and 1B currents exhibited slowed
activation and reduced amplitudes, reflecting inhibition of the
underlying Ca channels through a G-protein-dependent pathway. As
expected, the inhibited 1A or 1B channels
could be facilitated by a conditioning depolarization. Less pronounced
facilitation was also observed with GTP instead of GTPS in the
pipette solution (data not shown). In contrast, facilitation was absent
from cells dialyzed with GDPS.
Ca current amplitudes decreased during dialysis with GTPS.
After 5 min of whole-cell recording, 1A currents
had decreased to 59 ± 7% (n = 26 cells) of the
initial amplitude (recorded within 60 sec of establishing the
whole-cell configuration), and 1B currents had decreased
to 56 ± 6% (n = 35). These decreases probably reflect the onset of G-protein-dependent inhibition combined with Ca
channel run-down. By way of contrast, during 5 min dialysis with
GDPS, the amplitudes of 1A currents increased to
122 ± 7% (n = 9), and those of 1B
currents increased to 149 ± 7% (n = 10) of
initial amplitudes. These increases likely result from Ca current
run-up combined with removal of preexisting G-protein-dependent inhibition.
Facilitation of 1E
It is now well established that 1A and
1B are inhibited through G-protein-dependent pathways
(Herlitze et al., 1996; Toth et al., 1996; Zhang et al., 1996; Zamponi
et al., 1997). In contrast, whether 1E is also modulated
by the same pathways has been unclear. Some previous studies have
concluded that 1E is inhibited by G-proteins (Yassin et
al., 1996; Mehrke et al., 1997; Qin et al., 1997; Shekter et al.,
1997), whereas others have concluded that it is insensitive to
G-protein inhibition (Bourinet et al., 1996; Toth et al., 1996; Page et
al., 1997). To examine this issue further, we studied facilitation of
1E under the same experimental conditions as
1A and 1B.
Dialysis with GTPS decreased the amplitude of 1E
currents to 74 ± 1% (n = 18) of initial levels,
suggesting the development of G-protein-mediated inhibition. Consistent
with this interpretation, dialysis with GDPS increased
1E current amplitudes to 151 ± 12% of initial
levels (n = 7). 1E currents exhibited
significant kinetic slowing (Diverse-Peirluissi et al., 1995),
activating at +10 mV with an average time constant
(1) of 4.4 ± 0.3 msec in the presence of
intracellular GTPS (n = 18) compared with 2.9 ± 0.5 msec (n = 7) with internal GDPS. As
illustrated in Figure 2, kinetic slowing
of 1E currents could be reversed by a conditioning
depolarization, consistent with inhibition of 1E channels through a membrane-delimited pathway. Using the standard voltage protocol (as in Fig. 1), we observed a slight facilitation of
1E current amplitudes in some cells; one example is
illustrated in Figure 2A. However, in most cells
1E current amplitudes were not facilitated. In contrast,
nearly all cells dialyzed with GTPS exhibited significant
facilitation of activation kinetics. Facilitation of 1E
apparently requires G-protein activation, because it was absent from
cells dialyzed with GDPS.
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Figure 2.
G-protein-dependent facilitation of
1E. A, Facilitation of 1E
current amplitudes and activation kinetics in a cell dialyzed with
GTPS (left) but not in a cell dialyzed with GDPS
(right). Voltage protocol as in Figure 1.
Left, Data file 98403058; C = 32 pF;
RS = 2.8 M. Right, Data
file 97602030; C = 31 pF;
RS = 2.4 M. B,
Facilitation of 1E is greatly enhanced by shortening the
conditioning and test pulses. Left, 1E
currents evoked by the standard voltage protocol in which P1, P2, and
CP were each 25 msec in duration. Data file 98406202;
C = 17 pF; RS = 3.5 M. Right, 1E currents evoked in the
same cell by a briefer protocol to the same voltages but with P1 and P2
reduced to 10 msec and CP reduced to 12 msec in duration. Data file
98406204; C = 17 pF; RS = 3.5 M.
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We also observed that pronounced facilitation of 1E
current amplitudes could be produced by shortening the durations of the test and conditioning pulses (Fig. 2B). This
observation suggests that inactivation of 1E channels in
response to the standard voltage protocol usually obscured facilitation
of macroscopic current amplitudes.
Facilitation is correlated with current density
Cells transfected with 1A,
1B, or 1E expressed a wide range
of Ca current densities (from unmeasurable to ~400 pA/pF). To examine
whether the expression level of Ca channels might influence their
modulation by G-proteins, we plotted the magnitude of facilitation as a
function of the maximal current density in each cell (Fig. 3). Facilitation was quantified as the
ratio
I2/I1, in
which I2 is the peak current evoked by P2, and
I1 is the peak current evoked by P1 of the
standard voltage protocol (Fig. 1). As an additional measure of
facilitation we used the ratio
1/2, where 1
is the time constant for activation of
I1, and 2 is the time
constant for activation of I2. The plots in
Figure 3 reveal that facilitation of 1A,
1B, and 1E is negatively
correlated with current density. Thus, low-density currents exhibited
the most facilitation and high-density currents exhibited the least
facilitation.
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Figure 3.
The magnitude of facilitation is negatively
correlated with current density. The ratio
I2/I1
(left panels) or
1/2 (right panels)
is plotted as a function of maximal Ca current density for cells
expressing 1A (n = 26),
1B (n = 35), or 1E
(n = 37). I1 and
I2 are the amplitudes measured at the time
of peak inward Ca current evoked by P1 and P2, respectively, of the
standard voltage protocol (Fig. 1). P1 and P2 were to +30 mV (for
1A and 1B) or +10 mV (for
1E); facilitation was maximal at these voltages
(Figs. 4-6). The pipette solution contained GTPS. For the plots
shown, current densities were determined within 60 sec of establishing
the whole-cell configuration, and facilitation was calculated for
currents recorded after 5 min of whole-cell dialysis. The
lines are linear regressions; the p value
listed in each plot indicates the statistical significance of the
correlation coefficient. When current densities determined after >5
min of whole-cell dialysis were used as the independent variable, the
p values were 0.0008, 0.0001, and 0.0003 for
I2/I1
ratios and 0.27, 0.012, and 0.036 for
1/2 ratios of
1A, 1B, and
1E, respectively. All subsequent comparisons of
1A, 1B, and
1E used only currents having initial densities of  50
pA/pF (dashed vertical line).
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Voltage dependence of facilitation
We next compared the voltage dependence of facilitation for
1A, 1B, and
1E Ca channels. To minimize variability attributable to
differences in channel density, we restricted our analysis throughout
this study to data from cells expressing 1A,
1B, or 1E currents at initial
densities of 50 pA/pF (Fig. 3, vertical dashed
lines). As shown in Figure
4A, in cells dialyzed
with GTPS, the amplitudes of currents mediated by 1A
were significantly facilitated (i.e., I2
exceeded I1) at test potentials of +20, +30, +40, and +50 mV, whereas at more positive test potentials I2 and I1 were equal. In
contrast, the activation rates of 1A currents were
facilitated at all test potentials from +20 to +90 mV (Fig.
4B). Thus, 2 was significantly smaller
than 1 over the entire range of test potentials at which
these time constants could be reliably determined. For comparison, in
cells dialyzed with GDPS no facilitation of current amplitudes or
activation kinetics was observed at any test potential (Fig. 4).
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Figure 4.
Voltage dependence of facilitation for
1A. A, Current-voltage relationships
with GTPS or GDPS in the pipette solution.
I1 and I2 were
normalized to the maximal I1 recorded in
each cell and then averaged. B, Average values of
1 and 2; the time constants for
activation of I1 and
I2, respectively, are plotted as a
function of test potential. Data are from seven (GTPS) and four
(GDPS) cells. Voltage protocol as in Figure 1.
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Similar results were obtained for 1B (Fig.
5). However, a notable difference was
that 1B current amplitudes were facilitated over a
smaller range of test potentials (+20, +30, and +40 mV) than were found
for 1A. Thus, I2 was smaller than
I1 at voltages above +40 mV, presumably because
of inactivation of 1B channels in response to the
standard voltage protocol. In contrast, the activation rates of
1B currents were facilitated at all test potentials from
+20 to +80 mV. Thus, for both 1A and 1B
the activation rates of currents were facilitated over a much wider range of test potentials than were current amplitudes.
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Figure 5.
Voltage dependence of facilitation for
1B. Data from six (GTPS) and four (GDPS) cells.
Legend otherwise as in Figure 4.
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Using the standard voltage protocol, current amplitudes were
facilitated in only ~40% (7 of 18) of cells expressing
1E at initial current densities 50 pA/pF.
Consequently, the average values of I2 did not
exceed those of I1 (Fig.
6A). Nonetheless, the
average amplitudes of 1E currents clearly indicated the
presence of G-protein-dependent modulation, because there was a much
greater difference between I2 and
I1 in cells dialyzed with GDPS than in cells
dialyzed with GTPS (Fig. 6A). Furthermore, cells
dialyzed with GTPS exhibited kinetic slowing that was almost
completely reversed the conditioning pulse (Fig. 6B).
In contrast, kinetic slowing and facilitation were absent from cells
dialyzed with GDPS. Taken together with results presented in Figure
2, these data demonstrate G-protein-dependent inhibition and
facilitation of 1E Ca channels.
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Figure 6.
Voltage dependence of facilitation for
1E. Data from eight (GTPS) and six (GDPS) cells.
Legend otherwise as in Figure 4.
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No facilitation of 1C
In contrast to 1A,
1B, and 1E subunits, the cardiac
1C subunit was not affected by G-protein activation. In
cells dialyzed with GTPS, I2 was consistently
smaller than I1, presumably because of
Ca-dependent inactivation of 1C (Fig.
7). Also in contrast to
1A, 1B, and
1E, the voltage dependences of
I1 and I2 were not
appreciably different for 1C (Fig. 7B).
Neither was activation of 1C currents speeded by a
conditioning depolarization (Fig. 7C). Furthermore, the
amplitudes of 1C currents decreased less than
1A and 1B currents during dialysis with
GTPS (to 82 ± 8% of initial levels; n = 11),
and, unlike 1A, 1B, and
1E, the amplitudes of 1C currents
did not increase significantly during dialysis with GDPS (104 ± 5% of control, n = 4). In summary, we were unable
to detect any significant differences between 1C currents recorded with GTPS and those recorded with GDPS in the
pipette solution. These results are consistent with previous studies
(Bourinet et al., 1996; Toth et al., 1996; Zhang et al., 1996)
reporting that 1C is not inhibited by G-proteins.
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Figure 7.
Absence of G-protein-dependent facilitation of
1C. A, Representative 1C
currents recorded from a cell dialyzed with GTPS. Data file
97512011; C = 12 pF; RS = 3.5 M. B, Average voltage dependence of
I1 and I2;
data from three cells dialyzed with GTPS.
I1 and I2 were
normalized by the maximal I1 in each cell.
C, Average voltage dependence of 1 and
2; data from three cells dialyzed with GTPS.
Voltage protocol as in Figure 1.
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1A and 1B are facilitated to
similar degrees
Previous studies have concluded that 1B is more
strongly inhibited than 1A through G-protein-dependent
pathways and also that G-protein-inhibited 1B channels
are more strongly facilitated by a conditioning depolarization than
1A channels (Bourinet et al., 1996; Zhang et al., 1996;
Zamponi et al., 1997). These studies have used neurotransmitter
receptors to induce the phasic activation of G-proteins. To examine
whether 1B is also more strongly facilitated in the
presence of tonically activated G-proteins, we compared 1A and 1B currents in cells dialyzed with
GTPS. As shown in Figure
8A, the average
I2/I1 ratios of
1A and 1B currents were identical,
demonstrating that the amplitudes of 1A and
1B currents were facilitated to the same extent. Control
experiments with intracellular GDPS produced smaller
I2/I1 ratios for
1B (0.63 ± 0.03; n = 10) than for
1A (0.95 ± 0.02; n = 9),
suggesting that the voltage protocol caused greater inactivation of
unmodulated 1B channels than of unmodulated
1A channels.
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Figure 8.
Comparative facilitation of
1A, 1B,
1C, and 1E Ca channels.
A, Average
I2/I1
ratios for currents recorded with GTPS (filled
bars) or GDPS (unfilled bars) in the pipette.
Voltage protocol as in Figure 1. P1 and P2 were to +30 mV
(1A, 1B, and
1C) or +10 mV (1E).
B, Average 1/2
ratios for the same currents. In cells dialysed with GTPS, the
maximal current densities were 16 ± 3 pA/pF
(n = 23) for 1A, 15 ± 3 pA/pF (n = 23) for 1B,
16 ± 5 pA/pF (n = 11) for
1C, and 36 ± 4 pA/pF (n = 18) for 1E. In cells dialysed with GDPS, the
maximal current densities were 20 ± 4 pA/pF
(n = 9) for 1A, 12 ± 4 pA/pF (n = 10) for 1B, 6 ± 1 pA/pF (n = 4) for 1C,
and 28 ± 7 pA/pF (n = 7) for
1E.
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Figure 8B compares the facilitation of
1A and 1B activation kinetics. The
average 1/2 ratios of
1A and 1B currents were not significantly
different, indicating that the conditioning pulse speeded activation of
1A and 1B to the same degree. Thus, once
1A and 1B channels have been inhibited by
tonically activated G-proteins, they are equally facilitated by a
conditioning depolarization.
Figure 8 also presents data for 1E. With intracellular
GTPS, the average
I2/I1 ratio for
1E was 1.03 ± 0.03 (n = 18),
whereas with intracellular GDPS this ratio was only 0.61 ± 0.06 (n = 7), indicating a significant
(p < 0.001) G-protein-dependent effect. Further
evidence of modulation was provided by the substantial facilitation of
1E activation kinetics. In fact, the average 1/2 ratios for 1E
currents were statistically indistinguishable from those of
1A and 1B currents (Fig.
8B). Thus, with intracellular GTPS these
1/2 ratios were 1.79 ± 0.09 (n = 21), 1.60 ± 0.09 (n = 19),
and 1.55 ± 0.05 (n = 8) for
1A, 1B, and
1E, respectively (p = 0.07). With intracellular GDPS, these ratios were 1.09 ± 0.03 (n = 8), 1.03 ± 0.01 (n = 9), and
1.04 ± 0.03 (n = 7), respectively (p = 0.17). These comparisons further establish
the ability of 1E to be modulated through a
G-protein-dependent, presumably membrane-delimited pathway.
The kinetics of facilitation are very similar for
1A, 1B, and
1E
Facilitation is thought to reflect dissociation of G
subunits from Ca channels. Facilitation is transient and decays with time after a conditioning depolarization because G subunits rebind to channels and reestablish inhibition at negative potentials. To further explore the relative modulation of neuronal Ca channels by
tonically activated G-proteins, we compared both the onset and the
decay of facilitation for 1A,
1B, and 1E.
The onset of facilitation was measured by plotting
I2/I1 or
1/2 ratios of 1A
and 1B currents as a function of conditioning pulse
duration. For 1E we plotted only
1/2 ratios. As shown in Figure
9, the onset of facilitation could be
approximated by a single exponential function, producing a time
constant (onset) to describe this process. As the
duration of the conditioning pulse was increased from 0 to 30 msec, the
I2/I1 ratio
increased with a time constant of 4.18 ± 0.18 msec
(n = 5) for 1A currents and 5.30 ± 0.20 msec (n = 7) for 1B currents.
Although this difference is statistically significant
(p = 0.003), it is quite small. Similarly, 1/2 ratios increased with time
constants of 4.48 ± 0.96 msec (n = 5) for
1A currents, 3.87 ± 0.58 msec (n = 7) for 1B currents, and 3.76 ± 0.42 msec
(n = 9) for 1E currents; these time
constants are not different (p = 0.72). Thus,
facilitation develops with very similar kinetics for
1A, 1B, and
1E Ca channels.
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|
Figure 9.
Facilitation develops with similar time course for
1A, 1B, and
1E channels. 1/2
ratios (A) and
I2/I1
ratios (B) are plotted as a function of the
conditioning pulse (CP) duration for representative
cells. Plots were fit by single exponential functions to yield time
constants for the onset of facilitation (onset).
Average values of onset determined using
1/2 or
I2/I1
ratios are summarized graphically in the bottom right
corner. The pipette solution contained GTPS.
1A, Data file 98115076; C = 29 pF; RS = 2.4 M.
1B, Data file 98129067; C = 27 pF; RS = 3.8 M.
1E, Data file 98205005; C = 19 pF; RS = 3.2 M. Data from cells
expressing maximal current densities of 28 ± 5 pA/pF
(1A, n = 5), 21 ± 6 pA/pF (1B, n = 7), and 38 ± 4 pA/pF (1E,
n = 9). Voltage protocol as in Figure 1, except
that P1 and CP were separated by 50 msec at 90 mV. Each point is the
average of two currents.
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|
The decay of facilitation was monitored by plotting
I2/I1 or
1/2 ratios as a function of a
variable interval ( T) between the conditioning pulse and the second
test pulse (Fig. 10). Because the decay
of facilitation varies with its magnitude (Golard and Siegelbaum, 1993;
Elmslie and Jones, 1994), we only compared currents that were
facilitated to similar degrees
(I2/I1 ratios of
1.6 ± 0.1 for 1A and 1.7 ± 0.1 for
1B; and 1/2
ratios of 2.1 ± 0.2 for 1A, 1.8 ± 0.2 for 1B, and 1.6 ± 0.1 for
1E). The decays of
I2/I1 and
1/2 were fit by single exponential
functions, and time constants for reinhibition (reinhib)
were obtained.
I2/I1 ratios
decayed with an average time constant of 48 ± 8 msec
(n = 7) for 1A currents and 48 ± 3 msec (n = 6) for 1B currents (p = 0.94).
1/2 ratios decayed with an average
time constant of 51 ± 8 msec (n = 7) for
1A currents, 53 ± 10 msec (n = 6) for 1B currents, and 55 ± 7 msec
(n = 8) for 1E currents
(p = 0.94). These results indicate that
facilitation decays from 1A,
1B, and 1E Ca channels at very
similar speeds.
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Figure 10.
Facilitation decays from
1A, 1B, and
1E Ca channels at the same rate.
1/2 ratios
(A) and
I2/I1
ratios (B) are plotted as a function of T, the
variable interval between CP and P2 for representative cells. Each plot
was fit by a single exponential function to yield a time constant for
reinhibition (reinhib). Average values of
reinhib are summarized in the bar graphs (bottom
right). The pipette solution contained GTPS.
1A, Data file 97731092; C = 24 pF; RS = 2.9 M.
1B, Data file 97729010; C = 21 pF; RS = 3.0 M.
1E, Data file 97D24046; C = 16 pF; RS = 3.7 M. Data from cells
expressing maximal current densities of 16 ± 4 pA/pF
(1A, n = 7), 23 ± 6 pA/pF (1B, n = 6), and
39 ± 6 pA/pF (1E, n = 8).
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|
The kinetics of modulation can be represented by the scheme (after
Currie and Fox, 1997; Zhou et al., 1997):
During a facilitating depolarization to +100 mV, G subunits
should dissociate from the channels. If G subunits do not also
rebind channels during the depolarization, then
koff can be approximated by
1/onset. On repolarization to 90 mV, G subunits
should rebind to channels at a rate equal to kon
[G] + koff. If resting inhibition of Ca
channels by G subunits is strong, as suggested by the absence of
a separate, rapidly activating component of current (Fig. 1), then
koff should be small, and kon [G] can be approximated by
1/reinhib. Assuming that all three channel types
experience similar concentrations of G subunits, our estimates of
onset and reinhib suggest that
koff and kon have very
similar values for 1A, 1B,
and 1E. Although this argument is not rigorous, it is
consistent with the idea that G subunits dissociate from and
reassociate with 1A, 1B,
and 1E channels at very similar or identical rates.
Large segments of 1B are unnecessary for its
modulation by G-protein
It was previously demonstrated by Zhou et al. (1995) that deleting
large portions of the cytoplasmic II-III loop and C terminus from
lB (mutant lB-DD) does not
eliminate G-protein-dependent inhibition or facilitation. However, in
their experiments lB-DD was expressed in dysgenic
myotubes, where the magnitude of facilitation was small and where
kinetic slowing was not apparent in either wild-type lB
or mutant lB-DD currents, raising the possibility that
the native behavior of lB might not be fully reproduced within the cellular environment of skeletal muscle. To further examine
the functional importance of the II-III loop and C terminus in Ca
channel modulation, we expressed lB-DD in HEK293 cells and quantified its G-protein-dependent facilitation.
In lB-DD, amino acids 829-995 have been deleted
from the II-III loop, and residues 1877-2338 have been deleted from
the C terminus (Fig.
11A). As illustrated
in Figure 11B, currents mediated by
lB-DD exhibited strong facilitation of activation rates
and current amplitudes. The voltage dependences of inhibited and
facilitated lB-DD currents (I1
and I2, respectively) were very
similar (Fig. 11C) to currents mediated by the
full-length lB (Fig. 5), confirming that the basic
voltage-dependent properties of lB-DD were not appreciably changed by its deletions. During 5 min of intracellular dialysis with GTPS, the amplitudes of lB-DD currents
decreased to 63 ± 13% (n = 6) of initial levels,
comparable to the decrease observed for the full-length
lB (56 ± 6%, n = 35).
Interestingly, with intracellular GTPS the
I2/I1 ratio for
lB-DD was significantly larger than for wild-type
lB (2.18 ± 0.15, n = 6; vs
1.67 ± 0.08, n = 23; p = 0.01),
suggesting greater facilitation or perhaps less inactivation of the
mutant, although the voltage dependence of I2
suggests that inactivation of lB-DD was unaltered.
I2/I1 ratios (Fig.
11D) were also slightly larger for
lB-DD than for lB in the presence of
intracellular GDPS (0.86 ± 0.06, n = 5; vs
0.63 ± 0.03, n = 10; p = 0.001).
However, the activation kinetics of lB-DD and
lB currents were equally facilitated, and
1/2 ratios were indistinguishable
between wild-type and mutant channels (Fig. 11E).
Activation rates of lB-DD and lB currents
were also identical in the absence of G-protein stimulation. For
example, with intracellular GDPS and at a test potential of +30 mV,
1 was 3.1 ± 0.3 msec (n = 5) for
lB-DD and 3.1 ± 0.2 msec (n = 10)
for lB. These results confirm that amino acids 829-995
and 1877-2338 are not required for modulation of lB by
G-proteins and further demonstrate that these channel regions are not
needed for facilitation of current amplitudes or activation
kinetics.
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Figure 11.
Undiminished G-protein-dependent modulation of
1B-DD. A, Diagrammatic representation of
the mutant N-type Ca channel 1B-DD, which lacks
amino acids 829-995 from the II-III loop and amino acids 1877-2338
from the C terminus. The deleted regions are indicated by dashed
lines. B, Facilitation of 1B-DD
currents, evoked using the standard voltage protocol. Data file
97321108; C = 43 pF; RS = 3.9 M. C, Voltage dependence of inhibited
(I1) and facilitated
(I2) currents mediated by
1B-DD. I1 and
I2 were normalized to the maximal
I1 in each cell (n = 4).
The standard voltage protocol was used. D, Facilitation
of 1B-DD current amplitudes is slightly larger than for
wild-type 1B. Standard voltage protocol, with P1 and P2
to +30 mV. The pipette contained GTPS (filled
bars) or GDPS (unfilled bars).
E, Facilitation of activation kinetics is identical for
1B-DD and 1B. With intracellular GTPS,
1/2 ratios were 1.60 ± 0.09 (n = 21) for 1B and 1.56 ± 0.11 (n = 6) for 1B-DD
(p = 0.91). With intracellular GDPS,
1/2 ratios were 1.03 ± 0.01 (n = 5) for 1B and 1.06 ± 0.05 (n = 5) for 1B-DD
(p = 0.41). D,
E, Data from cells with maximal current densities of
15 ± 3 pA/pF (1B,
n = 23) and 18 ± 6 pA/pF
(1B-DD, n = 6) in the GTPS
experiments and 11 ± 5 pA/pF (1B,
n = 5) and 6 ± 1 pA/pF
(1B-DD, n = 5) in the GDPS
experiments.
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|
| |
DISCUSSION |
The magnitude of G-protein-dependent facilitation correlates with
Ca current density
Variations in channel density have previously been shown to
correlate with significant differences in channel behavior (cf. Adams
et al., 1996). In the present study we found that low-density 1A, 1B, and
1E currents exhibited the greatest amount of
facilitation, whereas high-density currents often lacked facilitation
(Fig. 3). This observation suggests that Ca channel density somehow influences the extent to which G-proteins are able to produce inhibition. A correlation between current density and extent of modulation might result if cells expressing a high density of Ca
channel 1 subunits also expressed an excess of Ca
channel subunits, which are thought to antagonize
G-protein-mediated Ca channel inhibition (Campbell et al., 1995) by
competing with G for binding sites on the 1
subunit (De Waard et al., 1997; Qin et al., 1997). Alternatively, cells
expressing high densities of Ca channels might express insufficient
G-proteins to inhibit all of the Ca channels, or the endogenous
G-proteins of HEK293 cells may not have access to Ca channels expressed
at high densities.
1A and 1B exhibit similar modulation
by tonically activated G-proteins
In our experiments, 1A and 1B showed
the same amount of G-protein-dependent facilitation. Thus,
I2/I1 ratios and
1/2 ratios were equivalent between
1A and 1B channels (Fig. 8). Furthermore, our measurements of onset and reinhib
(Fig. 9, 10) strongly suggest that G-proteins dissociate from and
reassociate with 1A and 1B Ca channels at
the same rates. Our findings that 1A and
1B are equally facilitated and also have the same
kinetics of facilitation differ from the results of previous studies
(Bourinet et al., 1996; Zhang et al., 1996; Currie and Fox, 1997;
Zamponi et al., 1997). Our results may arise from our expression of a
particular combination of Ca channel subunits, our consideration of Ca
current density in the data analysis, or our use of GTPS to produce
tonically activated as opposed to phasically activated G-proteins (but
see Currie and Fox, 1997 for differential effects of GTPS on native P/Q- and N-type channels). As one possibility, 1B
channels may be localized more closely to neurotransmitter receptors
and/or G-proteins than 1A channels, such that
1B would experience a higher concentration of G
subunits after receptor activation. In contrast, during tonic
activation of G-proteins with GTPS, the concentration of G may
be relatively uniform throughout the cell, and differences between the
localization of 1A and 1B might not
affect their modulation. This possibility is in keeping with the
conclusions of Wilding et al. (1995) and Zhou et al. (1997) that
activated opioid receptors can only inhibit nearby Ca channels,
suggesting that phasically activated G-proteins have a limited range.
It is also possible that GTPS does not activate as many G-proteins,
or the same varieties of G-proteins, as activated neurotransmitter
receptors.
1E exhibits G-protein-dependent inhibition
and facilitation
In previous studies, Bourinet et al. (1996), Toth et al., (1996),
and Page et al. (1997) concluded that 1E was not
significantly inhibited by G-proteins, whereas Yassin et al. (1996),
Mehrke et al. (1997), Shekter et al. (1997), and Qin et al. (1997)
concluded the opposite. Our present results confirm the latter view.
Although the amplitudes of 1E currents were not always
facilitated using the standard voltage protocol, we observed very
consistent facilitation of 1E activation kinetics (Figs.
2, 6, 8). In fact, the average 1/2
ratios of 1E currents were indistinguishable from those of 1A and 1B currents (Fig.
8B), suggesting that the magnitude of
G-protein-dependent facilitation is quite comparable among 1A, 1B, and
1E channels. Our results also indicate that
voltage-dependent inactivation can obscure facilitation of
1E current amplitudes. Inactivation produced a similar,
although less pronounced, effect on the facilitation of
1B current amplitudes (Fig. 5). Thus, activation
kinetics appear to be more sensitive and more reliable than current
amplitudes as an index of G-protein-dependent modulation, particularly
for channels such as 1E that inactivate at relatively negative potentials.
On the macroscopic level, Ca channel facilitation can be manifested as
increased current amplitudes, increased activation rates, or both.
Inactivation of some Ca channels can prevent facilitation of
macroscopic current amplitudes but should not prevent the facilitated opening (i.e., shorter first latency; Patil et al., 1996) of the remaining noninactivated channels. Thus, facilitation of activation kinetics should occur even if the facilitating depolarizations inactivate some channels. Under physiological conditions in which Ca
channel activation and subsequent Ca influx is triggered by action
potentials and other brief depolarizations, a facilitation of Ca
channel activation kinetics may be more functionally significant than a
facilitation of current amplitudes. Ca channels formed by
1E appear to be localized to neuronal cell bodies and
dendrites (Yokoyama et al., 1995). The ability of 1E to
undergo G-protein-mediated inhibition and facilitation may therefore be
important for the integration and propagation of dendritic electrical
signals and possibly also for gene transcription within neuronal cell
nuclei.
1C does not exhibit
G-protein-dependent facilitation
The rabbit cardiac 1C subunit did not exhibit
kinetic slowing or facilitation in the presence of tonically activated
G-proteins. Our results thus agree with previous reports (Bourinet et
al., 1994, 1996; Zhou et al., 1995; Zhang et al., 1996) that
1C is not modulated through a membrane-delimited,
G-protein-dependent pathway. In our experiments 1C also
did not exhibit voltage-dependent facilitation, in contrast to results
obtained with the rat neuronal 1C expressed in
Xenopus oocytes (Bourinet et al., 1994; Cens et al., 1996).
Voltage-dependent facilitation may be limited to particular splice
variants of 1C, manifested only in certain expression systems, or may require longer conditioning depolarizations than used in the present study.
A G interaction site identified within the C terminus of
1E is not required for modulation of
1B
We quantified the G-protein-dependent facilitation of a deletion
mutant of the N-type Ca channel (lB-DD) that is
missing amino acids 829-995 from the II-III loop and amino acids
1877-2338 from the C terminus (Fig. 11A).
lB-DD displayed the same magnitudes of kinetic slowing
and facilitation of activation kinetics as the full-length, wild-type
lB subunit (Fig. 11D,E), demonstrating that the missing regions are not required for G-protein-dependent inhibition or facilitation of lB. This result is
particularly significant in view of the recent identification by Qin et
al. (1997) of an ~38 amino acid residue sequence within the C
terminus of lE that is required for its inhibition by
G. The corresponding region of lB, which
was shown by Qin et al. (1997) to bind G in vitro, is
not present within the 1B-DD mutant. Therefore, this
region cannot also be essential for G-protein-dependent modulation of
1B. The results obtained with 1B-DD thus
raise the intriguing possibility that different structural regions of
1B and 1E mediate their interactions with
G-proteins.
| |
FOOTNOTES |
Received Oct. 30, 1997; revised April 29, 1998; accepted May 4, 1998.
This work was supported by National Institutes of Health Grant R01
NS34423 and a Basic Research Grant from the Muscular Dystrophy Association to B. Adams. U.M. was the recipient of a Consejo Nacional de Ciencia y Tecnologia Fellowship. We thank K. Campbell, Y. Mori, T. Snutch, and T. Tanabe for providing expression plasmids, T. Smith for
technical assistance, and two anonymous reviewers for constructive
criticisms.
Correspondence should be addressed to Dr. Brett Adams, Department of
Physiology and Biophysics, 5-660 Bowen Science Building, University of
Iowa College of Medicine, Iowa City, IA 52242-1109.
| |
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Top
Abstract
Introduction
