 |
 Previous Article | Next Article 
Volume 16, Number 15,
Issue of August 1, 1996
pp. 4617-4624
Copyright ©1996 Society for Neuroscience
Selective G-Protein Regulation of Neuronal Calcium Channels
Peter T. Toth1,
Lee R. Shekter1,
Gloria Hui
Ma1,
Louis H. Philipson2, and
Richard J. Miller1
1 Department of Pharmacological and Physiological
Sciences, and 2 Department of Medicine, The University of
Chicago, Chicago, Illinois 60637
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
We examined the properties and regulation of Ca channels resulting
from the expression of human  1B and
1E subunits stably expressed in HEK293 cells.
The ancillary subunits  1B and
2/ were also stably expressed in these cell
lines. Ca currents in 1B-expressing cells had
the properties of N-type currents. Ca currents in cells expressing
1E exhibited a novel profile that was similar
to the properties of the ``R type'' Ca current. Introduction of
GTP- -S into 1B cells greatly enhanced the
extent of prepulse facilitation of the Ca current, whereas it had only
a very small effect in 1E-expressing cells.
Activation of somatostatin receptors endogenous to HEK293 cells or  opioid receptors, expressed in the cells after transfection, inhibited
Ca currents in 1B-expressing cells. This
inhibition was blocked by pertussis toxin and was partially relieved by
a depolarizing prepulse. In contrast, no inhibitory effects were noted
in cells expressing 1E channels under the same
circumstances. HEK293 cells normally contained G-proteins from all of
the four major families. Inhibition of Ca currents by agonists in
1B-expressing cells was enhanced slightly by
the cotransfection of several G-protein subunits. agonists,
however, had no effect in 1E-containing cells,
even after overexpression of different G-protein -subunits. In
summary, these results demonstrate that there is a large difference in
the susceptibility of 1B- and
1E-based Ca channels to regulation by
G-proteins. This is so despite the fact that the two types of Ca
channels show substantial similarities in their primary sequences.
Key words:
Ca channel;
G-protein;
receptor;
patch
clamp;
HEK293;
somatostatin
INTRODUCTION
Regulation by receptors, G-proteins, and second
messengers is one of the most widely observed characteristics of Ca
channels (Hille, 1994 ; Dolphin, 1995). For example, the activation of L
channels in the heart by cAMP-dependent phosphorylation has been
studied extensively, and the physiological significance of these
observations is clear (Perez-Reyes et al., 1994). In the nervous
system, it has been shown frequently that activation of various
G-protein-linked receptors can produce inhibition of N channels (Hille,
1994). Because it is known that N channels play a critical role in the
regulation of transmitter release at many synapses (Miller, 1990;
Hille, 1994), this process is believed to be a key element in the
receptor regulation of synaptic communication, particularly the
phenomenon of presynaptic inhibition. Rather than being mediated by a
diffusible second messenger, ``rapid'' inhibition of N channels is
thought to be attributable to direct effects of G-protein subunits on
some part of the Ca channel complex (Hille, 1994). Therefore, this type
of N-channel regulation has been described as ``membrane delimited.''
It is not yet clear, however, which subunits of the G-protein
heterotrimer actually produce these effects or to which part of the N
channel they bind (Sternweis, 1994; Wickman and Clapham, 1995).
N channels, which result from the expression of the
1B Ca channel subunit, are found primarily in
neurons and neuroendocrine cells (Williams et al., 1992; Wheeler et
al., 1995). This is also the case for at least two other types of Ca
channels: the P/Q type, which probably results from the expression of
1A, and the recently described
1E subunit, which may produce a Ca current of
the ``R'' type (Williams et al., 1994; Wheeler et al., 1995).
Interestingly, the 1A,
1B, and 1E subunits
form a Ca channel subfamily and exhibit a high degree of sequence
homology (Dolphin, 1995; Wheeler et al., 1995). None of these Ca
channels are sensitive to dihydropyridines, which block
1C, 1D, and
1S Ca channels and to which they exhibit less
sequence homology (Dolphin, 1995; Wheeler et al., 1995). It is now
clear that Ca currents of the P/Q type, which can also support
neurotransmitter release (Wheeler et al., 1994), are regulated by
G-proteins and receptors in a manner similar to N channels (Mintz and
Bean, 1993; Rhim and Miller, 1994; Kanemasa et al., 1995). It is not
known at this point, however, whether Ca currents resulting from the
expression of 1E are also regulated in this
way. Indeed, very little is known about the properties and biological
functions of Ca channels resulting from 1E
expression. We have now investigated this question and demonstrate that
there seem to be considerable differences in the ability of
1B and 1E to be
regulated by G-proteins.
MATERIALS AND METHODS
HEK293 cell lines. HEK293 cell lines expressing Ca
channels with various subunit compositions were kindly provided by
SIBIA Inc. (Williams et al., 1992; Bleakman et al., 1995). Briefly, the
cell lines were developed by stable co-transfection of HEK293 cells
with human 1, 2B,
and Ca channel subunit expression plasmids. The G1A1 cell line
consisted of
1B-1-2B-1B
subunits. The subunit composition of the A4A2 cells was
1B-1-2B-1C.
The E52-3 cell line expressed the 1E-type Ca
channel with the
1E-3-2B-1B
subunit composition.
HEK293 cells stably expressing Ca2+-channels were
grown in plastic Falcon dishes in DMEM (Life Technologies,
Gaithersburg, MD) containing 5% defined bovine serum (HyClone, Logan,
UT) plus penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml),
and geneticin (500 µg/ml). One day before recording, cells were
dissociated by gentle trituration with a fire-polished Pasteur pipette
and replated onto poly-L-lysine-coated glass
coverslips.
Preparation of G-protein expression plasmids. cDNAs encoding
subunits of Gi1-3 and
Go (kindly provided by Randall R. Reed, Johns
Hopkins University) were subcloned into the mammalian expression vector
pCMV5 (Andersson et al., 1989) and confirmed by DNA sequencing using a
modification of the dideoxy-chain termination method (Sequenase 2.0;
USB, Cleveland, OH). The cDNA Gs was similarly
subcloned into pCMV6b and confirmed by sequencing (Robishaw et al.,
1986). The cDNAs encoding G13 and
Gq in pcDNA3 were obtained from G. Babnigg
(University of Chicago). opioid receptor cDNA in pCMV5 was a kind
gift of Graeme Bell (University of Chicago).
Transfection of HEK293 cells. Monolayers of HEK293 cells not
exceeding 75% confluence were dissociated and replated onto
poly-L-lysine-coated (Sigma, St. Louis, MO) glass
coverslips. Cells were cotransfected with plasmids containing the cDNAs
for the receptor, G-protein, and -galactosidase using the
standard calcium-phosphate precipitation technique (Ausubel et al.,
1993) or transfection kit (Mammalian Transfection Kit; Stratagene, La
Jolla, CA).
Analysis of gene and cDNA expression. For patch-clamp
experiments, duplicate coverslips were routinely stained for
-galactosidase expression, with an average transfection efficiency
of 40-70% determined by cell counting. Currents were recorded 48-72
hr after transfection.
The expression of the receptor was detected using the specific
antibody (kindly provided by Robert Elde, University of Minnesota) and
ABC kit (Vector Laboratories, Burlingame, CA).
The expression of the G-protein subunits was evaluated using
PCR-Southern blot and Northern blot analysis, as follows. Total RNA was
prepared using the CsCl guanidinium isothiocyanate method (Sambrook et
al., 1989). Poly(A+) RNA was prepared using an
oligo-dT-Sepharose column or an mRNA purification kit (Pharmacia,
Uppsala, Sweden); 15-20 µg of total RNA or 1-2 µg of
poly(A+) RNA was loaded per lane. RNA was
transferred to Hybond-N+ (Amersham) by overnight
blotting. The blots were hybridized using the
32P-labeled-specific
oligonucleotide probes (internal sequence) for the individual G-protein
subunits as shown below. In some control experiments (not shown),
cDNAs for specific G-proteins were 32P-labeled by
random priming and also used as probes. After hybridization [in the
solution containing 20% formamide, 5× SSC (1× SSC: 150 mM NaCl, 15 mM sodium
citrate), 2.5× Denhardt's solution] for 12-20 hr, the membranes
were rinsed in 2× SSC, 0.1% SDS for 10 min at room temperature,
washed in 0.5× SSC, 0.1% SDS at 42°C for 20-40 min, and then
exposed to Kodak X-omat film for 12 hr.
For PCR-based detection of expression, first-strand cDNA was prepared
from total RNA using random primers and Superscript reverse
transcriptase (Life Technologies, Grand Island, NY). The following
oligonucleotide primer pairs (forward and reverse) were used.
Gi1 Forward: 5 -CTGTGGAAGGACAGCGGTGTG-3
Reverse: 5-CAGCAACAGAGAATGTAGTG-3
Internal: 5-GCAGTGGGGTAGTAAAAATGCATT-3
Gi2Forward: 5-CCTGTCGGGCGTCATCCGGAG-3
Reverse: 5-CCATGCTCCCTGCCTGTTCCC-3
Internal: 5-GATGAATCGCATGCATGAGAGC-3
Gi3Forward: 5-GATTAAACGTTTATGGCGAG-3
Reverse: 5-GCATGACAGGACCAAGGAATG-3
Internal: 5-GAGGATGGCATAGTAAAAGCT-3
GoForward: 5-CACTGAACCATTCTCTGCAG-3
Reverse: 5-TTTGGCCTTTGTAAGACACAC-3
Internal: 5-CACTCAGCGGCTATGACCAG-3
GsForward: 5-GACCAACCGCCTGCAGGAGGC-3
Reverse: 5-GGGCATGATTAACAAAGCAACC-3 (bovine)
Reverse: 5-GGGCATGATTCACACCGCAACC-3 (rat)
Internal: 5-CACGCAGTTGATCACCCACC-3
GqForward: 5-CTACCCCTGGTTCCAGAACTCC-3
Reverse: 5-CACGCTCACAGAGTCCAGGACG-3
Internal: 5-GTCGACTAGGTGGGAATACATG-3
G13Forward: 5-CGGGTTTTCAGCAACGTCTCC-3
Reverse: 5-TCAGCAGCTGTCAGCCACA-3
Internal: 5-CTCTTAAGCAGTGGGGGTCC-3
The PCR was used under the following conditions: 45 sec at
94°C, 1 min at 55°C, and 1 min at 72°C for 30 cycles, followed by
10 min at 72°C. An aliquot of the PCR sample was analyzed by
electrophoresis in 1% agarose. DNAs were transferred to
Hybond-N+ (Amersham) and then hybridized with the
specific oligonucleotide probe (internal) for each G-protein subunit shown above, using the conditions described.
Whole-cell patch clamp. The tight-seal whole-cell
configuration of the patch-clamp technique (Hamill et al., 1981) was
used to record Ca currents. Recordings were made at room temperature
(21-24°C). Currents were recorded using an Axopatch 1D (Axon
Instruments, Foster City, CA) amplifier, filtered at 2 kHz by the
built-in filter of the amplifier, and stored on the computer.
Capacitative transients were canceled at 10 MHz, and their values were
obtained directly, together with the series-resistance values from the
settings of the Axopatch 1D amplifier. Series-resistance compensation
between 40 and 80% was applied. Leak corrections were performed using
a P/N protocol. Command pulses were delivered at 20 sec intervals.
Soft, soda-lime capillary glass was used to make patch pipettes, which
were coated with Sylgard (Dow Corning, Midland, MI) and had resistances
of 1.8-3.5 M when filled with internal solution. Extracellular
buffer solution for whole-cell voltage-clamp experiments was composed
of (in mM): 160 tetraethylammonium chloride, 5 CaCl, 1 MgCl2, 10 HEPES, 10 glucose; pH was
adjusted to 7.4 with TEAOH. The standard internal solution consisted of
(in mM): 100 CsCl, 37 CsOH, 1 MgCl2, 10 BAPTA, 10 HEPES, 3.6 MgATP, 1 GTP, and
14 Tris2CP, and 50 U/ml-1
CPK. The pH was adjusted to 7.3 with CsOH. The osmolarity of the
pipette solution was 300 mOsm/l, and the osmolarity of the
extracellular solution was between 315 and 323 mOsm/l. GTP was replaced
by GTP--S or by GDPS in the double-pulse and tail-current
experiments.
Stock solutions of U69593 (RBI, Natick, MA) and nor-BNI (RBI) were
prepared in ethanol and stored at  20°C.
[D-Trp8] somatostatin
(SOM) stock solutions (Bachem, Belmont, CA) were kept at 80°C.
Stock solutions of PTX (100 µg/ml; RBI) were prepared in water and
stored at 4°C. Cells were treated with PTX at a final concentration
of 200 ng/ml overnight.
RESULTS
Effects of GTP analogs
We examined the regulation of 1B-
and 1E-based Ca channels stably expressed in
HEK293 cells. Ca channels in these cells also contained stably
expressed 2/ and
1B subunits (Williams et al., 1992; Bleakman
et al., 1995). It has been shown that the presence of these ancillary
subunits is essential for Ca channels to be expressed efficiently and
display all of their normal properties (Williams et al., 1992; Bleakman
et al., 1995). Ca currents could be elicited in these cells by step
depolarizations to different potentials. Cells expressing
1B produced Ca currents that had properties of
typical N currents (Bleakman et al., 1995). Cells expressing
1E produced currents whose properties we have
described previously and which are similar to currents described in the
literature as ``R'' type (Williams et al., 1994; Wheeler et al.,
1995).
We wished to compare the regulation of 1B and
1E channels by G-proteins. To do this, we
began by examining the effect of activating the G-proteins normally
found in HEK293 cells directly, using the nonhydrolyzable analog of
GTP, GTP--S. One interesting phenomenon was apparent immediately.
Normally there was a marked increase in the amplitude of the Ca current
in 1B-expressing cells in the period after the
pipette broke into the cell (Fig.
1A). This ``run-up'' of the current
was not apparent in GTP--S-containing cells (Fig. 1B).
One of the hallmarks of rapid ``membrane-delimited'' N-current
inhibition is that it can be relieved by a depolarizing prepulse to
high voltages (Bean, 1989; Hille, 1994). If the current observed during
GTP--S perfusion represents an inhibited current, then a
depolarizing prepulse should increase its amplitude. We therefore used
a voltage-clamp protocol that consisted of two test pulses with or
without a strong intervening depolarization to +80 mV (Fig.
1C,D). The ratio of current integrals (P2/P1) was plotted as
a function of time (Fig. 2A-C). The
ratio P2/P1 became larger after the prepulse (Fig.
2A). Figure 2A shows that the
maximum GTP--S effect developed ~4 min after establishing
whole-cell recording conditions. At this time, there was no difference
in P2/P1 ratios with GDPS in the pipette (Fig. 2B),
consistent with the ``inhibitory'' action of GDPS on G-protein
function. The small difference in the P2/P1 ratios observed using 1 mM GTP in the patch pipette was presumably
attributable to a small degree of basal G-protein activation under
these conditions (Fig. 2C). Interestingly, the degree of
``run-up'' in 1E-expressing cells was less
marked than in 1B-expressing cells. Although
there was a trend suggesting that this small degree of run-up was also
suppressed by GTP--S, this was not significant (Fig. 1E).
Furthermore, in these cells, the current evoked by a test pulse after a
depolarization also was actually smaller than without the prepulse
(Fig. 1F). This was presumably attributable to differences
in the extent of voltage-dependent inactivation between the two types
of Ca channels (Williams et al., 1994; Bleakman et al., 1995).
Consistent with this finding, the ``R''-type current in cerebellar
granule neurons also displayed very pronounced voltage-dependent
inactivation (Zhang et al., 1993). Thus, for
1E currents, the value of the P2/P1 ratio
after the prepulse fell below that seen without the prepulse (Fig.
2D-F). We therefore normalized the data by subtracting out
the degree of voltage-dependent inactivation. This was obtained from
cells perfused with GDPS, in which we assumed that no
G-protein-induced inhibition would be present. After this
transformation, the data obtained with GTP or GTP--S in the pipette
followed a trend similar to that obtained with
1B-based channels (Fig. 2D-F,
red data sets). Comparison of the data in Figure
2A-C with that in D-F, however, suggests that
although there is some G-protein-mediated inhibition of
1E channels, this is 7- to 10-fold less than
that obtained with 1B channels. This suggests
that there is a considerable difference in the ability of G-proteins to
regulate 1B- and
1E-based Ca channels directly.
Fig. 1.
Ca currents in HEK293 cells expressing the
1B and 1E Ca channel
subunits. A, Run-up with intracellular GTP using Ca (5 mM) as the charge carrier in HEK293 cells
expressing the 1B Ca channel subunit. Currents
were evoked from a holding potential of 90 mV by 200 msec
depolarizing pulses to +10 mV every 20 sec. B, Average
normalized current with 1 mM GTP ( ) or 0.3 mM GTP--S ( ) in the patch pipette. The
run-up of currents from individual experiments was normalized with
respect to the first peak current obtained. The normalized values were
then averaged, and the mean ± SEM was plotted (1 mM GTP, n = 11; 0.3 mM GTP--S, n = 6). C,
D, Relief of GTP--S-induced inhibition of
1B Ca currents by an intervening prepulse
depolarization. Ca currents were evoked using a double-pulse protocol
without (C, lower trace) or with (D,
lower trace) a depolarizing prepulse using GTP--S (0.3 mM) in the patch pipette. The intervening
depolarization increased the current amplitude during the second pulse
(D, lower trace). Upper lines in
C and D are the voltage templates (HP = 90
mV; TP = +10 mV; TP duration = 25 msec; prepulse
depolarization potential = +80 mV; duration = 50 msec). P1
and P2 denote the current integrals during the first and second test
pulses and are used as such in Figure 2. E, Characteristics
of Ca current run-up in cells expressing 1E
subunit. Plot of averaged Ca current amplitude (mean ± SEM) in
the presence of GTP--S (, 0.3 mM;
n = 15) or GTP (, 1 mM;
n = 6). Calculations as in B. F,
Superimposed Ca2+ current traces evoked by the
double-pulse voltage protocol with or without a depolarizing prepulse
(1 mM GTP in the patch pipette). The Ca current
following the prepulse depolarization was actually smaller than without
it in the cell line expressing 1E
subunit.
[View Larger Version of this Image (25K GIF file)]
Fig. 2.
Comparison of the effect of different GTP analogs
on the 1B (A-C) and
1E (D-F) Ca current using the
double-pulse protocol (see Fig. 1C,D). Currents were evoked
every 20 sec in HEK293 cells expressing the 1B
subunit by applying the double-pulse voltage protocol with GTP--S
(A), GDPS (B), or GTP (C) in the
patch pipette. The P2/P1 ratios from individual cells were calculated,
and then the isochronal values were averaged during the time course of
the experiments from cells in which experiments were carried out under
identical conditions. The mean ± SE is plotted; denotes the
P2/P1 ratio without the prepulse, denotes the ratio with the
prepulse. GTP analogs were applied in the following concentrations:
GTP--S (0.3 mM; n = 5);
GDPS (0.3 mM; n = 7); GTP (1 mM; n = 11). D-F,
Effect of GTP analogs on 1E Ca currents using
the double-pulse protocol. Plot of the average P2/P1 ratios in the
presence of GTP--S, 0.3 mM (n = 15; D), GDPS, 0.3 mM
(n = 7; E), and GTP, 1 mM (n = 6; F). Note
that the values of P2/P1 ratios after prepulse application () fell
below those of P2/P1 values obtained without the prepulse
depolarization () (black data sets on D-F).
D-F, red data sets show P2/P1 ratios with
prepulse after ``subtraction'' of voltage-dependent inactivation
obtained from data with GDPS in the patch pipette.
[View Larger Version of this Image (30K GIF file)]
Receptor regulation of Ca currents
The results presented above demonstrate that direct activation of
G-proteins in these cell lines produces strong inhibition of
1B but not 1E
channels. G-proteins are normally activated by ``serpentine''
receptors, and this can lead to inhibition of Ca currents in neurons.
We attempted to reconstitute receptor regulation of Ca channels in
HEK293 cells using endogenous and exogenous receptors. There are
several examples in the literature of the regulation of Ca currents by
somatostatin receptors (Ikeda and Schofield, 1989; Golard and
Siegelbaum, 1993; Fujii et al., 1994; Hille, 1994), and endogenous
somatostatin receptors have been reported to exist in HEK293 cells (Law
et al., 1993). We found that activation of these receptors with the
somatostatin analog SOM produced substantial inhibition of the Ca
currents in 1B-expressing cells (Fig.
3A,D). Little or no inhibition, however, was
observed in cells expressing 1E channels (Fig.
3C,D). Inhibition of the 1B Ca
current by SOM was repeatable, exhibiting modest desensitization (38.5 ± 5.5% inhibition on first application, 26.2 ± 3.8% inhibition on
second; n = 6). Furthermore, inhibition of
1B currents was blocked by pretreatment of
cells with PTX (Fig. 3D) and was relieved partially by a
depolarizing prepulse (Fig. 3B). We also observed that SOM
was equally effective in inhibiting the Ca current in a second
1B-expressing cell line that differed in the
type of subunit (1C) expressed (Fig.
3D).
Fig. 3.
Effect of SOM on 1B-type
and 1E Ca currents. A, Plot of
1B-type Ca current versus time showing a
typical SOM (300 nM) response. Cell was
depolarized from 90 HP to +10 TP every 20 sec. Inset shows
Ca currents recorded before and during SOM application. B
shows 1B-type Ca currents evoked by the
double-pulse protocol in the presence of SOM (300 nM). Decreased inhibition can be seen after the
prepulse. C, Plot of 1E-type Ca
current versus time: SOM had little or no effect on the Ca current.
D, Average responses (mean ± SEM) to SOM (300 nM) application. The number in
parentheses represents the number of experiments.
[View Larger Version of this Image (19K GIF file)]
Activation of opioid receptors in some neurons has also been shown
to produce inhibition of Ca currents (Lipscombe et al., 1989; Rhim and
Miller, 1994). In contrast to the effects of SOM, however, addition of
a selective agonist for the opioid receptor U69593 produced no
inhibition of Ca currents in either the 1B or
1E cell line. This was consistent with the
lack of opioid receptors in HEK293 cells as indicated by
immunostaining or Northern blot analysis (data not shown). We therefore
transiently transfected the opioid receptor into both
1B- and 1E-expressing
cell lines. Northern blots and immunostaining indicated similar degrees
of transfection into the two cell lines (not shown). After
transfection, U69593 robustly inhibited the Ca current in
1B- but not in
1E-containing cells (Fig. 4).
Inhibition by SOM was retained in these cells (data not shown).
U69593-induced inhibition was blocked by nor-BNI, a specific inhibitor
of opioid receptors (Fig. 4). Inhibition produced by U69593 was
also partially voltage-dependent and inhibited by PTX treatment (Figs.
4, 5).
Fig. 4.
Effects of the receptor-selective agonist
U69593 on receptor-transfected HEK293 cells expressing
1B and 1E Ca
channels. Average responses (mean ± SEM) to U69593 (200 nM); n = number of cells showing
response to receptor agonist application (left to right, bars 1, 3, and 4) or all cells tested (bars 2 and 5). In experiments in which the
blocking effect of nor-BNI was examined, cells were also transfected
with the subunit of Go (see below). In the
experiments examining the blocking effect of PTX (200 ng/ml overnight),
cells were also transfected with the -subunit of
Gi2 (see text).
[View Larger Version of this Image (22K GIF file)]
Fig. 5.
Effect of G-protein subunit overexpression on
Ca current inhibition by receptor activation in cells expressing
1B Ca channels. A-C, Plots of Ca
current versus time. Insets show Ca current traces at the
points indicated before and during the application of the receptor
agonist U69593 (200 nM). HEK293 cells were
transfected with only the receptor (A), receptor + Gi1 (B), receptor + Go (C), and receptor + Gi3 (D). D, Superimposed
control and inhibited (U69593; 200 nM) Ca current
traces from a cell expressing the 1B Ca
channel, receptor, and Gi3. The U69593
inhibition was partially relieved by a prepulse depolarization (see
Fig. 2).
[View Larger Version of this Image (18K GIF file)]
It seemed possible that our inability to observe strong G-protein
regulation of 1E currents was attributable to
the fact that HEK293 cells lacked a particular G-protein that was
required for regulation of 1E. We observed,
however, that under normal conditions the cells expressed mRNAs for
many different G-proteins, including representative members of all of
the four major families (i/o,
s, q,
13) (data not shown). Nevertheless, it was
still possible that the actual quantities of some of these might be
limiting. We therefore overexpressed several different G-protein subunits with and without the opioid receptor and examined the
ability of U69593 or SOM to inhibit Ca currents in the two cell lines.
Overexpression of each of the G-protein subunits was confirmed by
Northern blot analysis. Overexpression of some of the G-protein subunits actually slightly enhanced the ability of U69593 to inhibit Ca
currents in 1B-expressing cells (Figs.
5B-D, 6A),
although the effects of SOM were not altered (Fig. 6B). In
cells overexpressing G-protein subunits, the effects observed were
still blocked by PTX (Figs. 4, 6A) and were still partially
voltage-dependent (Fig. 5D). On the other hand, no
substantial effect of U69593 (Fig. 6C) on
1E currents was observed, even in cells in
which we overexpressed different G-protein subunits.
Fig. 6.
A, Average inhibition (mean ± SEM) of 1B Ca currents by U69593 (200 nM) in HEK293 cells expressing
1B Ca channels, different G-protein subunits, and the opioid receptor. n denotes the number
of cells showing agonist responses, except the first and last bars,
where all of the responses were averaged. B, Inhibitory
effects (mean ± SEM) of SOM (300 nM) in
HEK293 cells expressing 1B Ca channels, receptors, and various G-protein subunits. n denotes the
number of responsive cells. C, Average inhibition (mean ± SEM) of the Ca current by U69593 (200 nM) in
HEK293 cells expressing 1E Ca channels, receptors, and different G-protein subunits. n = total number of cells.
[View Larger Version of this Image (21K GIF file)]
DISCUSSION
We have used an HEK293 cell expression system to make some initial
attempts at understanding the G-protein regulation of Ca channels. The
two types of Ca channels investigated in the present series of
experiments, 1B and
1E, are both members of the family of
nondihydropyridine-sensitive channels generally found in neurons and
neuroendocrine cells. These subunits exhibit a high degree of
homology with each other; they are ~80% homologous in those regions
that have been suggested as compromising the 24 transmembrane helices.
The degree of homology between the two types of channels falls to
~40%, however, in nontransmembrane-spanning regions such as the
intracellular loop connecting domains 3 and 4 and the intracellular
C-terminal extension (Soong et al., 1993; Schneider et al., 1994;
Williams et al., 1994; Wheeler et al., 1995). Considering the high
degree of overall homology between 1B and
1E and of these proteins with
1A, it is of considerable interest to note the
large difference in the ability of the two channel types to be
regulated by G-proteins. The regulation of N channels that we have
observed in HEK293 cells closely resembles that frequently described in
neurons (Hille, 1994). Little is known as yet, however, about the
normal properties and functions of the Ca channels that are formed by
expression of 1E, although the protein is
widely expressed on the soma and dendrites of neurons throughout the
brain (Williams et al., 1994; Volsen et al., 1995; Yokoyama et al.,
1995). The experiments reported here suggest that direct G-protein
regulation of these channels may be quite minimal. It should be noted
that in our studies we found that G-proteins representative of all of
the four major families were represented in HEK293 cells, and at least
one member of each of these families was also overexpressed in our
experiments. Thus, although it remains possible that
1E channels are normally regulated through an
exceedingly restricted direct G-protein pathway that we have missed,
this seems unlikely. It should be noted in this regard that although N
channel inhibition tends to be mediated by PTX-sensitive G-proteins,
there are several examples of non-PTX-sensitive G-proteins also
producing this type of direct, membrane-delimited inhibition (Hille,
1994; Zhu and Ikeda, 1994). This implies that for N channels, many
types of G-protein subunits may be able to interact with the
channel. Another interpretation of these observations would be that it
is actually the / subunits that produce the inhibition and that
these may be released from various receptor-activated G-protein
heterotrimers (Ikeda, 1996; Herlitze et al., 1996). Whichever mechanism
is involved, it seems unlikely that any G-protein regulation of highly
homologous 1E channels would be highly
selective. In our experiments, we have also tried to produce regulation
of 1E channels in several different ways.
Thus, introduction of GTP--S into cells would activate the entire
complement of HEK293 cell G-proteins (Zhou et al., 1995; Zong et al.,
1995). Furthermore, we also used somatostatin receptors (Law et al.,
1993; Shapiro and Hille, 1993; Reisine and Bell, 1995) and opioid
receptors (Shen and Crain, 1994; Avidor-Reiss et al., 1995; Ikeda et
al., 1995; Lai et al., 1995; Tallent et al., 1995), both of which can
activate a wide variety of G-proteins. Interestingly, all of these
manipulations produced robust inhibition of 1B
channels, which had the same ancillary subunit composition as the
1E channels. We conclude that it is unlikely
that we failed to observe regulation of 1E
channels because of the fact that we failed to express the correct
components for observing such regulation, if it did indeed exist.
The characteristics of inhibition of 1B
channels observed in the present experiments are similar in many
respects to those reported previously. For example, the inhibition
displayed both voltage-dependent and -independent components (Bean,
1989; Hille, 1994; Diverse-Pierluissi and Dunlap, 1995;
Diverse-Pierluissi et al., 1995). Whether this is attributable to
activation of more than one G-protein-linked pathway, thereby producing
more than one effect on the channels as suggested in the literature, is
difficult to assess (Diverse-Pierluissi and Dunlap, 1995;
Diverse-Pierluissi et al., 1995). Thus, HEK293 cells normally contained
various G-proteins, even before the introduction of more of these
molecules by transfection. Furthermore, both somatostatin and opioid receptors potentially can activate several of these G-proteins.
The results, however, are also consistent with a single process. The
effect of the depolarizing prepulse is assumed to lower the affinity of
the interaction between the G-protein subunit(s) and the channel that
allows unbinding (Boland and Bean, 1993). It is possible that rebinding
of the G-protein might occur with a time course that is beyond the
resolution of this study.
The present observations set some limitations on the
localization of G-protein-binding to the 1B
channel. Thus, there is a high degree of sequence identity between
1B and 1E in their 24 putative membrane-spanning domains (Soong et al., 1993; Schneider et
al., 1994; Williams et al., 1994; Wheeler et al., 1995). On the other
hand, other areas exhibit less homology. This is particularly true of
the large intracellular loop between domains 2 and 3 and the N and C
termini. Furthermore, the smaller intracellular loop between domains 1 and 2 also shows considerable divergence. It is interesting to note
that this small loop is the site of interaction between the Ca channel
subunit and the small subunit (Pragnell et al., 1994). Some
studies have suggested that Ca channel subunits and G-protein
subunits may compete for a binding site on the Ca channel
1 subunit, although it is not clear whether
such an interaction is competitive or allosteric (Berrow et al., 1995;
Campbell et al., 1995b; Roche et al., 1995). It has been suggested
further that the subunits of Ca channels may possess GAP activity
(Campbell et al., 1995a) and that the Ca channel and subunits
cooperate in enhancing the GTPase activity of the G-protein subunit. Such observations are interesting in light of recent studies
showing that G-protein and / subunits both bind to GIRK-1,
one of the G-protein-regulated K channels (Huang et al., 1995). These
studies also suggested that GIRK-1 may also possess GAP activity
(Slesinger et al., 1995). It is possible that a similar arrangement
also applies to Ca channels. Resolution of this problem will require
elucidation of precisely which G-protein subunits bind to and regulate
Ca channels.
FOOTNOTES
Received Feb. 15, 1996; revised April 18, 1996; accepted May 2, 1996.
This work was supported by Public Health Service Grants DA02121,
MH40165, DA02575, and NS33502. We thank Drs. Michael Harpold (SIBIA
Inc.) and David Lodge (Eli Lilly) for providing the cell lines and for
helpful discussions. We thank Dongjun Ren for her technical help.
Correspondence should be addressed to Richard J. Miller, Department of
Pharmacological and Physiological Sciences, The University of Chicago,
947 East 58th Street (MC 0926), Chicago, IL 60637.
REFERENCES
-
Andersson S,
Davis DL,
Dahlback H,
Jornvall H,
Russell DW
(1989)
Cloning, structure and expression of the
mitochondrial cytochrome P-450 sterol 25-hydroxylase, a bile acid
biosynthetic enzyme.
J Biol Chem
264:8222-8229 .
[Abstract/Free Full Text]
-
Ausubel FM,
Brent R,
Kingston RE,
Moore DD,
Seidman JG,
Smith JA,
Struhl K
(1993)
Transfection of DNA into eukaryotic cells.
In: Current protocols in molecular biology,
, p. 9.1.1. New York: Wiley.
-
Avidor-Reiss T,
Zippel R,
Levy R,
Saya D,
Ezra V,
Barg J,
Matus-Leibovitch N,
Vogel Z
(1995)
-opioid receptor
transfected cell lines: modulation of adenylyl cyclase activity
following acute and chronic opioid treatment.
FEBS Lett
361:70-74 .
[Web of Science][Medline]
-
Bean BP
(1989)
Neurotransmitter inhibition of neuronal
calcium channels by changes in channel voltage dependence.
Nature
340:153-156 .
[Medline]
-
Berrow NS,
Campbell V,
Fitzgerald EM,
Brickley K,
Dolphin AC
(1995)
Antisense depletion of subunits modulates the
biophysical and pharmacological properties of neuronal calcium
channels.
J Physiol (Lond)
482:481-491 .
[Abstract/Free Full Text]
-
Bleakman D,
Bowman D,
Bath CP,
Brust PF,
Johnson EC,
Deal CR,
Miller RJ,
Ellis SB,
Harpold MM,
Hans M,
Grantham CJ
(1995)
Characteristics of a human N type calcium channel
expressed in HEK293 cells.
Neuropharmacology
34:753-765 .
[Web of Science][Medline]
-
Boland LM,
Bean BP
(1993)
Modulation of N type calcium
channels in bullfrog sympathetic neurons by luteinizing hormone
releasing hormone, kinetics and voltage dependence.
J Neurosci
13:516-533 .
[Abstract]
-
Campbell V,
Berrow N,
Brickley K,
Page K,
Wade R,
Dolphin AC
(1995a)
Voltage dependent calcium channel subunits in
combination with 1 subunits have a GTPase
activating effect to promote the hydrolysis of GTP by
Gao in rat frontal cortex.
FEBS Lett
370:135-140 .
[Web of Science][Medline]
-
Campbell V,
Berrow EM,
Fitzgerald EM,
Brickley K,
Dolphin AC
(1995b)
Inhibition of the interaction of G-protein
Go with calcium channels by the calcium channel
subunit in rat neurons.
J Physiol (Lond)
485:365-372 .
[Abstract/Free Full Text]
-
Diverse-Pierluissi M,
Dunlap K
(1995)
Interaction of
convergent pathways that inhibit N type calcium currents in sensory
neurons.
Neuroscience
65:477-483 .
[Web of Science][Medline]
-
Diverse-Pierluissi M,
Goldsmith PK,
Dunlap K
(1995)
Transmitter mediated inhibition of N type calcium
channels in sensory neurons involves multiple GTP binding proteins and
subunits.
Neuron
14:191-200 .
[Web of Science][Medline]
-
Dolphin AC
(1995)
Voltage dependent calcium channels and
their modulation by neurotransmitters and G-proteins.
Exp Physiol
80:1-36 .
[Web of Science][Medline]
-
Ewald DA,
Pang IH,
Sternweis PC,
Miller RJ
(1989)
Differential G protein-mediated coupling of
neurotransmitter receptors to Ca2+ channels in
rat dorsal root ganglion neurons in vitro.
Neuron
2:1185-1193 .
[Web of Science][Medline]
-
Fujii Y,
Gonoi T,
Yamada T,
Chihara K,
Inagaki N,
Seino S
(1994)
Somatostatin receptor subtype SSTR2 mediates the
inhibition of high voltage activated calcium channels by somatostatin
and its analogue SMS 201-995.
FEBS Lett
355:117-120 .
[Web of Science][Medline]
-
Golard A,
Siegelbaum SA
(1993)
Kinetic basis for the
voltage-dependent inhibition of N-type calcium current by somatostatin
and norepinephrine in chick sympathetic neurons.
J Neurosci
13:3884-3894 .
[Abstract]
-
Hamill OP,
Marty A,
Neher E,
Sakmann B,
Sigworth FJ
(1981)
Improved patch-clamp techniques for high resolution
current recording from cells and cell-free membrane patches.
Pflügers Arch
391:85-100 .
[Web of Science][Medline]
-
Herlitze S,
Garcia DE,
Mackie K,
Hille B,
Scheuer T,
Catterall WA
(1996)
Modulation of Ca2+ channels
by G-protein subunits.
Nature
380:258-262.
[Medline]
-
Hille B
(1994)
Modulation of ion channels by G-protein
coupled receptors.
Trends Neurosci
17:531-535 .
[Web of Science][Medline]
-
Huang CL,
Slesinger PA,
Casey PJ,
Jan YN,
Jan LY
(1995)
Evidence that direct binding of G to the
GIRK-1 G-protein gated inwardly rectifying K channel is important for
channel activation.
Neuron
15:1133-1143 .
[Web of Science][Medline]
-
Ikeda SR
(1996)
Voltage-dependent modulation of N-type
calcium channels by G-protein subunits.
Nature
380:255-258.
[Medline]
-
Ikeda SR,
Schofield GG
(1989)
Somatostatin blocks a calcium
current in rat sympathetic neurons.
J Physiol (Lond)
409:221-240 .
[Abstract/Free Full Text]
-
Ikeda K,
Kobayashi T,
Ichikawa T,
Usui H,
Kumanishi T
(1995)
Functional couplings of the and the opioid
receptors with the G-protein activated K channel.
Biochem Biophys Res Commun
208:302-308 .
[Web of Science][Medline]
-
Kanemasa T,
Asakura K,
Ninomiya M
(1995)
-opioid agonist
U50488 inhibits P type Ca channels by two mechanisms.
Brain Res
702:207-212.
[Web of Science][Medline]
-
Lai HWL,
Minami M,
Satoh M,
Wong YH
(1995)
Gz coupling to the rat
-opioid receptor.
FEBS Lett
360:97-99.
[Web of Science][Medline]
-
Law SF,
Yasuda K,
Bell GI,
Reisine T
(1993)
Gi3 and
Go selectively associate with the cloned
somatostatin receptor subtype SSTR2.
J Biol Chem
268:10721-10727 .
[Abstract/Free Full Text]
-
Lipscombe D,
Kongsamut S,
Tsien RW
(1989)
Alpha-adrenergic
inhibition of sympathetic neurotransmitter release mediated by
modulation of N-type calcium-channel gating.
Nature
340:639-642 .
[Medline]
-
Miller RJ
(1990)
The receptor mediated regulation of calcium
channels and neurotransmitter release.
FASEB J
4:3291-3300 .
[Abstract]
-
Mintz IM,
Bean BP
(1993)
GABA-B receptor inhibition of P type
Ca channels in central neurons.
Neuron
10:889-998 .
[Web of Science][Medline]
-
Perez-Reyes E,
Yuan W,
Wei X,
Bers DM
(1994)
Regulation of
cloned L-type cardiac calcium channels by cyclic AMP dependent protein
kinase.
FEBS Lett
342:119-123 .
[Web of Science][Medline]
-
Pragnell M,
De Waard M,
Mori Y,
Tanabe T,
Snutch TP,
Campbell KP
(1994)
Calcium channel subunit binds to a conserved
motif in the 1-11 cytoplasmic linker of the 1
subunit.
Nature
368:67-70 .
[Medline]
-
Reisine T,
Bell G
(1995)
Molecular properties of somatostatin
receptors.
Neuroscience
67:777-790 .
[Web of Science][Medline]
-
Rhim H,
Miller RJ
(1994)
Opioid receptors modulate diverse
types of calcium channels in the nucleus tractus solitarius of the rat.
J Neurosci
14:7608-7615 .
[Abstract]
-
Robishaw JD,
Russell DW,
Harris BA,
Smigel MD,
Gilman AG
(1986)
Deduced primary structure of the -subunit of the
GTP-binding stimulatory protein of adenylate cyclase.
Proc Natl Acad Sci USA
83:1251-1255 .
[Abstract/Free Full Text]
-
Roche JP,
Anantharam V,
Treistman SN
(1995)
Abolition of
G-protein inhibition of 1A and
1B calcium channels by coexpression of the
3 subunit.
FEBS Lett
371:43-46 .
[Web of Science][Medline]
-
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular cloning:
a laboratory manual.
.
-
Schneider T,
Wei X,
Olcese R,
Costantin JL,
Neely A,
Palade P,
Perez-Reyes E,
Qin N,
Zhou J,
Crawford GD,
Smith RG,
Appel SH,
Stefani E,
Birnbaumer L
(1994)
Molecular analysis and functional
expression of the human type E neuronal Ca channel
1 subunit.
Recept Channels
2:255-270 .
[Web of Science][Medline]
-
Shapiro MS,
Hille B
(1993)
Substance P and somatostatin
inhibit calcium channels in rat sympathetic neurons via different G
protein pathways.
Neuron
10:11-20 .
[Web of Science][Medline]
-
Shen KF,
Crain SM
(1994)
Nerve growth factor rapidly prolongs
the action potential of mature sensory ganglion neurons in culture, and
this effect requires activation of Gs coupled
excitatory -opioid receptors on these cells.
J Neurosci
14:5570-5579 .
[Abstract]
-
Slesinger PA,
Reuveny E,
Jan YN,
Jan LY
(1995)
Identification
of structural elements involved in G-protein gating of the GIRK-1
potassium channel.
Neuron
15:1145-1156 .
[Web of Science][Medline]
-
Soong TW,
Stea A,
Hodson CD,
Dubel SJ,
Vincent SR,
Snutch TP
(1993)
Structure and functional expression of a member of
the low voltage activated calcium channel family.
Science
260:1133-1136 .
[Abstract/Free Full Text]
-
Sternweis PC
(1994)
The active role of / by in signal
transduction.
Curr Opin Cell Biol
6:198-203 .
[Web of Science][Medline]
-
Tallent M,
Dichter MA,
Bell GI,
Reisine T
(1995)
The cloned
kappa opioid receptor couples to an N-type calcium current in
undifferentiated PC-12 cells.
Neuroscience
63:1033-1040 .
-
Volsen SG,
Day NC,
McCormack AC,
Smith W,
Craig PJ,
Beattie R,
Incep G,
Show PJ,
Ellis SB,
Gillespie A,
Harpold MM,
Lodge D
(1995)
The expression of neuronal voltage dependent Ca
channels in human cerebellum.
Mol Brain Res
34:217-232.
-
Wheeler DB,
Randall A,
Tsien RW
(1994)
Roles of N type and Q
type Ca channels in supporting hippocampal synaptic transmission.
Science
264:107-111 .
[Abstract/Free Full Text]
-
Wheeler DB,
Randall A,
Sather WA,
Tsien RW
(1995)
Neuronal calcium channels encoded by the
1A subunit and their contribution to
excitatory synaptic transmission in the CNS.
Prog Brain Res
105:65-78 .
[Web of Science][Medline]
-
Wickman K,
Clapham DE
(1995)
Ion channel regulation by
G-proteins.
Physiol Rev
75:865-885 .
[Abstract/Free Full Text]
-
Williams ME,
Brust PF,
Feldman DH,
Patthi S,
Simerson S,
Maroufi A,
McCue AF,
Velicelebi G,
Ellis SB,
Harpold MM
(1992)
Structure
and functional expression of an
 -conotoxin sensitive human N-type
calcium channel.
Science
257:389-395 .
[Abstract/Free Full Text]
-
Williams SM,
Marubio LM,
Deal CR,
Hans M,
Brust PF,
Philipson LH,
Miller RJ,
Johnson EC,
Harpold MM,
Ellis SB
(1994)
Structure
and functional characterization of neuronal 1E
calcium channels subtypes.
J Biol Chem
269:22347-22357.
[Abstract/Free Full Text]
-
Yokoyama CT,
Westenbroek RE,
Hell JW,
Soong T-W,
Snutch TP,
Catterall WA
(1995)
Biochemical properties and subcellular distribution
of the neuronal class E calcium channel 1
subunit.
J Neurosci
15:6419-6432 .
[Abstract/Free Full Text]
-
Zhang J-F,
Randall AD,
Ellinor PT,
Horne WA,
Sather WA,
Tanabe Schwarz TL,
Tsien RW
(1993)
Distinctive pharmacology and kinetics of
cloned neuronal Ca2+ channels and their possible
counterparts in mammalian CNS neurons.
Neuropharmacology
32:1075-1088 .
[Web of Science][Medline]
-
Zhou J,
Zong S,
Tanabe T
(1995)
Modulation of cloned neuronal
calcium channels through membrane delimited pathway.
Biochem Biophys Res Commun
208:485-491 .
[Web of Science][Medline]
-
Zhu Y,
Ikeda SR
(1994)
VIP inhibits N type Ca channels of
sympathetic neurons via a pertussis toxin insensitive but cholera toxin
sensitive pathway.
Neuron
13:657-669 .
[Web of Science][Medline]
-
Zong S,
Yassin M,
Tanabe T
(1995)
G-protein modulation of
1A (P/Q) type calcium channel expressed in GH3
cells.
Biochem Biophys Res Commun
215:302-308 .
[Web of Science][Medline]
This article has been cited by other articles:
|
|
|
|
 
H. W. Tedford and G. W. Zamponi
Direct G Protein Modulation of Cav2 Calcium Channels
Pharmacol. Rev.,
December 1, 2006;
58(4):
837 - 862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Li, H. Zhong, T. Scheuer, and W. A. Catterall
Functional Role of a C-Terminal G{beta}{gamma}-Binding Domain of Cav2.2 Channels
Mol. Pharmacol.,
September 1, 2004;
66(3):
761 - 769.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Tan, M. Groszer, A. M. Tan, A. Pandya, X. Liu, and C.-W. Xie
Phosphoinositide 3-Kinase Cascade Facilitates {micro}-Opioid Desensitization in Sensory Neurons by Altering G-Protein-Effector Interactions
J. Neurosci.,
November 12, 2003;
23(32):
10292 - 10301.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. C. Foehring, P. G. Mermelstein, W.-J. Song, S. Ulrich, and D. J. Surmeier
Unique Properties of R-Type Calcium Currents in Neocortical and Neostriatal Neurons
J Neurophysiol,
November 1, 2000;
84(5):
2225 - 2236.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. R. Mirotznik, X. Zheng, and E. F. Stanley
G-Protein Types Involved in Calcium Channel Inhibition at a Presynaptic Nerve Terminal
J. Neurosci.,
October 15, 2000;
20(20):
7614 - 7621.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. A. Simen and R. J. Miller
Involvement of Regions in Domain I in the Opioid Receptor Sensitivity of alpha 1B Ca2+ Channels
Mol. Pharmacol.,
May 1, 2000;
57(5):
1064 - 1074.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. J. Kamp, H. Hu, and E. Marban
Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires beta -subunit
Am J Physiol Heart Circ Physiol,
January 1, 2000;
278(1):
H126 - H136.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Canti, K. M. Page, G. J. Stephens, and A. C. Dolphin
Identification of Residues in the N Terminus of alpha 1B Critical for Inhibition of the Voltage-Dependent Calcium Channel by Gbeta gamma
J. Neurosci.,
August 15, 1999;
19(16):
6855 - 6864.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Sun and R. J. Miller
Multiple Neuropeptide Y Receptors Regulate K+ and Ca2+ Channels in Acutely Isolated Neurons From the Rat Arcuate Nucleus
J Neurophysiol,
March 1, 1999;
81(3):
1391 - 1403.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Tanaka, I. Shibuya, N. Kabashima, Y. Ueta, and H. Yamashita
Inhibition of Voltage-Dependent Calcium Channels by Prostaglandin E2 in Rat Melanotrophs
Endocrinology,
December 1, 1998;
139(12):
4801 - 4810.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Meza and B. Adams
G-Protein-Dependent Facilitation of Neuronal alpha 1A, alpha 1B, and alpha 1E Ca Channels
J. Neurosci.,
July 15, 1998;
18(14):
5240 - 5252.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Page, C. Canti, G. J. Stephens, N. S. Berrow, and A. C. Dolphin
Identification of the Amino Terminus of Neuronal Ca2+ Channel alpha 1 Subunits alpha 1B and alpha 1E as an Essential Determinant of G-Protein Modulation
J. Neurosci.,
July 1, 1998;
18(13):
4815 - 4824.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Harayama, I. Shibuya, K. Tanaka, N. Kabashima, Y. Ueta, and H. Yamashita
Inhibition of N- and P/Q-type calcium channels by postsynaptic GABAB receptor activation in rat supraoptic neurones
J. Physiol.,
June 1, 1998;
509(2):
371 - 383.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. A. Simen and R. J. Miller
Structural Features Determining Differential Receptor Regulation of Neuronal Ca Channels
J. Neurosci.,
May 15, 1998;
18(10):
3689 - 3698.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. J Stephens, N. L Brice, N. S Berrow, and A. C Dolphin
Facilitation of rabbit {alpha}1B calcium channels: involvement of endogenous G{beta}{gamma} subunits
J. Physiol.,
May 15, 1998;
509(1):
15 - 27.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Zhang and J. T. Schmidt
Adenosine A1 Receptors Mediate Retinotectal Presynaptic Inhibition: Uncoupling by C-Kinase and Role in LTP During Regeneration
J Neurophysiol,
February 1, 1998;
79(2):
501 - 510.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Sun, L. H. Philipson, and R. J. Miller
Regulation of K+ and Ca++ Channels by a Family of Neuropeptide Y Receptors
J. Pharmacol. Exp. Ther.,
February 1, 1998;
284(2):
625 - 632.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B. A. McCool, J.-P. Pin, M. M. Harpold, P. F. Brust, K. A. Stauderman, and D. M. Lovinger
Rat group I Metabotropic Glutamate Receptors Inhibit Neuronal Ca2+ Channels via Multiple Signal Transduction Pathways in HEK 293 Cells
J Neurophysiol,
January 1, 1998;
79(1):
379 - 391.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C Dolphin
Mechanisms of modulation of voltage-dependent calcium channels by G proteins
J. Physiol.,
January 1, 1998;
506(1):
3 - 11.
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Chen and N. A. Lambert
Inhibition of Dendritic Calcium Influx by Activation of G-Protein-Coupled Receptors in the Hippocampus
J Neurophysiol,
December 1, 1997;
78(6):
3484 - 3488.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-W. Jeong and R. D. Wurster
Muscarinic Receptor Activation Modulates Ca2+ Channels in Rat Intracardiac Neurons via a PTX- and Voltage-Sensitive Pathway
J Neurophysiol,
September 1, 1997;
78(3):
1476 - 1490.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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):
282 - 291.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Boehm and H. Betz
Somatostatin Inhibits Excitatory Transmission at Rat Hippocampal Synapses via Presynaptic Receptors
J. Neurosci.,
June 1, 1997;
17(11):
4066 - 4075.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Page, G. J. Stephens, N. S. Berrow, and A. C. Dolphin
The Intracellular Loop between Domains I and II of the B-Type Calcium Channel Confers Aspects of G-Protein Sensitivity to the E-Type Calcium Channel
J. Neurosci.,
February 15, 1997;
17(4):
1330 - 1338.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Compound via MeSH
