Adenovirus-mediated Expression of an Olfactory Cyclic
Nucleotide-gated Channel Regulates the Endogenous
Ca2+-inhibitable Adenylyl Cyclase in C6-2B Glioma
Cells*
Kent A.
Fagan
,
Thomas C.
Rich§,
Shawna
Tolman¶,
Jerome
Schaack¶,
Jeffrey W.
Karpen§, and
Dermot M. F.
Cooper
From the Departments of
Pharmacology,
§ Physiology and Biophysics, and ¶ Microbiology,
University of Colorado Health Sciences Center,
Denver, Colorado 80262
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ABSTRACT |
Previous studies have established that
Ca2+-sensitive adenylyl cyclases, whether
endogenously or heterologously expressed, are preferentially regulated
by capacitative Ca2+ entry, compared with other means of
elevating cytosolic Ca2+ (Chiono, M., Mahey, R., Tate, G.,
and Cooper, D. M. F. (1995) J. Biol. Chem.
270, 1149-1155; Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444;
Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998)
J. Biol. Chem. 273, 9297-9305). These findings led to
the suggestion that adenylyl cyclases and capacitative Ca2+
entry channels were localized in the same functional domain of the
plasma membrane. In the present study, we have asked whether a
heterologously expressed Ca2+-permeable channel could
regulate the Ca2+-inhibitable adenylyl cyclase of C6-2B
glioma cells. The cDNA coding for the rat olfactory cyclic
nucleotide-gated channel was inserted into an adenovirus construct to
achieve high levels of expression. Electrophysiological measurements
confirmed the preservation of the properties of the expressed olfactory
channel. Stimulation of the channel with cGMP analogs yielded a robust
elevation in cytosolic Ca2+, which was associated with an
inhibition of cAMP accumulation, comparable with that elicited by
capacitative Ca2+ entry. These findings not only extend the
means whereby Ca2+-sensitive adenylyl cyclases may be
regulated, they also suggest that in tissues where they co-exist,
cyclic nucleotide-gated channels and Ca2+-sensitive
adenylyl cyclases may reciprocally modulate each other's activity.
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INTRODUCTION |
Cation-permeable, cyclic nucleotide-gated channels (CNG
channels)1 have traditionally
been considered in terms of their roles in visual and olfactory signal
transduction. The retinal channel, which is activated by cGMP, is
responsible for the "dark" current (1, 2), while the closely
related olfactory channel is gated by cAMP, and is thought to lead to
activation of Ca2+-activated Cl
currents and
membrane depolarization (3). Increasingly, however, a more widespread
function for these channels in cell physiology has been envisioned,
partially due to the finding that the channels are expressed in a wide
range of tissues and cell types. For instance, proteins homologous to
the CNG channel have been cloned from such diverse tissues as heart,
kidney, and testis, as well as from liver and skeletal muscle (4-6).
CNG channels have also been found in various brain regions, namely, the
hippocampus, cortex, and Purkinje cells of the cerebellum and other
neural derived tissues such as pineal and pituitary gland (5, 7-10).
The observation that these channels are widely expressed prompts a
reevaluation of their role in signal transduction. Functionally, the
CNG channels belong to the family of ligand-gated channels, but,
structurally, they are similar to voltage-gated channels. CNG channels
also share the important feature of Ca2+ permeation with
voltage-gated Ca2+ channels. At physiological
[Ca2+], an expressed, homomeric version of the olfactory
CNG channel exhibits a nearly "pure" Ca2+ current (11).
In comparison, only ~5% of the current through the NMDA channel is
carried by Ca2+ (12). Therefore, these channels provide a
second messenger-regulated form of Ca2+ entry into the cell
whose primary function may be to elevate [Ca2+]i.
Adenylyl cyclases are regulated by physiological transitions in
[Ca2+]i (reviewed in Refs. 13 and 14)). In fact,
of the nine currently described isoforms of adenylyl cyclase,
Ca2+ directly regulates four. Adenylyl cyclase types I and
VIII are stimulated, while types V and VI are inhibited by
submicromolar [Ca2+]. We have previously shown that
Ca2+-sensitive adenylyl cyclases are regulated by
capacitative Ca2+ entry (CCE) while they are refractory to
[Ca2+]i rises produced by other means, such as
release from internal stores or entry mediated by ionophore in
nonexcitable cells (15, 16). The dependence of these adenylyl cyclases on Ca2+ entering through CCE channels suggested a
functional colocalization of CCE channels and
Ca2+-sensitive adenylyl cyclases. Therefore, it was of
interest to determine whether Ca2+ entry through
heterologously expressed CNG channels might regulate these enzymes.
C6-2B cells, which endogenously express a Ca2+-inhibitable
adenylyl cyclase (type VI) (17), were used to determine whether
Ca2+ entry through an olfactory CNG channel could regulate
cAMP accumulation. Expression of the rat olfactory CNG channel (18) was
accomplished by creating an adenovirus construct containing the
channel. Infection with the adenovirus/CNG channel permits efficient
expression in a large majority of the cells. The expression of the
channel was evaluated by both [Ca2+]i
measurements in cell populations and electrophysiological methods.
Activation of the CNG channel with the cell-permeant cGMP analog,
CPT-cGMP, generated a [Ca2+]i rise that was
dependent on [CPT-cGMP], time of exposure to CPT-cGMP, and
[Ca2+]ex. Furthermore, activation of the
channel with CPT-cGMP and its associated [Ca2+]i
rise produced a substantial inhibition of cAMP accumulation. The
magnitude of the global [Ca2+]i rise generated by
the CNG channel was modest in comparison with capacitative
Ca2+ entry, but both modes of Ca2+ entry were
equally efficacious in their ability to reduce cAMP levels. Therefore,
Ca2+ entry through a heterologously expressed CNG channel
can modulate endogenous cAMP levels. These data not only show that a
Ca2+-sensitive adenylyl cyclase can be regulated by a
heterologously expressed Ca2+ channel, but also, that CNG
channels may play a role in modulating cAMP accumulation in tissues
where channels and adenylyl cyclases are co-expressed.
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EXPERIMENTAL PROCEDURES |
Materials--
Thapsigargin, forskolin, and Ro 20-1724 were from
Calbiochem. [2-3H]Adenine and [
-32P]ATP
were obtained from Amersham Pharmacia Biotech. Fura-2/AM and pluronic
acid were from Molecular Probes, Inc. (Eugene, OR). Other reagents were
from Sigma.
Cell Culture--
C6-2B rat glioma cells were maintained in 13 ml of F-10 medium (Life Technologies, Inc.) with 10% (v/v) bovine calf
serum (Gemini) in 75-cm2 flasks at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. Cells were plated at
approximately 70% confluency in 100-mm culture dishes for infection
with the CNG channel construct. 48 h postinfection, cells were
detached with phosphate-buffered saline containing 0.03% EDTA and
immediately used for measurement of cAMP accumulation or
[Ca2+]i.
Construction of Adenovirus Encoding the CNG Channel (See Fig.
1)--
A fragment encoding the rat
olfactory CNG channel
-subunit cDNA (18) was ligated between the
BamHI and SalI sites in the plasmid pACCMV, which
encodes the left end of the adenovirus chromosome with the E1A gene and
the 5'-half of the E1B gene replaced by the cytomegalovirus major
immediate early promoter, a multiple cloning site, and intron and
polyadenylation sequences from SV40 (19) to yield the plasmid
pACCMV-CNGC. pACCMV-CNGC was digested with SalI and ligated
with a BstBI adaptor in order to create pACCMV-CNGCBst, such that sequences encoding CNGC and the
left end of the adenovirus chromosome could be ligated directly to the
right arm of the adenovirus chromosome to create a transducing vector
using a newly developed
protocol.2
pACCMV-CNGCBst was digested with BstBI (to
provide an end to ligate with adenovirus DNA) and XmnI (to
provide a blunt end that would inhibit recircularization of the plasmid
as well as the formation of concatamers). The digested plasmid DNA was
ligated with BstBI-digested
Ad5dl327Bst
-Gal-TP complex (20).
Ad5dl327Bst
-Gal has a deletion of the
fragment between the XbaI sites at 28,593 and 30,471 base
pairs and therefore does not encode any of the products of the E3
region. The ligated DNA was used to transfect 293 cells using
Ca3(PO4)2 precipitation (21). The
transfected cells were incubated for 7 days. A freeze-thaw lysate was
prepared from the cells, and dilutions were used to infect 293 plates
for plaque purification. The infected 293 plates were overlaid with medium in Noble agar, fed after 4 days, and stained with
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside and
neutral red (20). Clear plaques, which are derived from recombination
that results in deletion of the lacZ gene present in the
parental viral chromosome, were amplified and analyzed by polymerase
chain reaction and restriction digestion for the presence of the CNGC
cDNA. Plaques that proved positive by polymerase chain reaction and
restriction digestion analysis were tested for the ability to direct
expression of CNGC. This procedure resulted in efficient production of
viruses encoding CNGC. The virus, termed Ad5dl327CMV-CNGC,
was grown in large scale, purified by successive banding on step and
isopycnic CsCl gradients, and dialyzed versus three changes
of 10 mM Tris-HCl, pH. 7.9, 135 mM NaCl, 1 mM MgCl2, 50% glycerol at 4 °C. Virus
particle concentration was quantitated by determination of the
absorbance at 260 nm. Virus was stored at
20° until use.

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Fig. 1.
Construction of
Ad5dl327CMV-CNGC. The
Ad5dl327Bst -Gal-TP complex (see "Experimental
Procedures") is represented at the top, with the
thick line indicating the foreshortened
adenovirus chromosome and TP indicated by filled in circles
at either end of the chromosome. The inverted terminal repeats
(ITR), which act as origins of replication, and the
cis-acting packaging sequence (PKG) are
indicated. The pACCMV-CNGC plasmid is indicated immediately
below the Ad5dl327Bst -Gal-TP complex.
Adenovirus sequence and the CMV-CNGC cassette are indicated by
thick lines, and plasmid vector (of which only
the ends associated with adenovirus sequence are shown) is indicated by
thin lines. Ad5dlBst- -Gal and
pACCMV-CMV-CNGC were restriction-digested as indicated, the restriction
digests were ligated together, and the ligated sample was used to
transfect HEK 293 cells. A lysate of the transfected cells was used to
infect new 293 cells, and plaques that were clear in the presence of
5-bromo-4-chloro-3-indolyl -D-galactopyranoside were
examined for the presence of the CNGC gene. The recombinant viral
chromosome encoding CNGC, Ad5dl327CMV-CNGC, is schematically
represented at the bottom.
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Measurement of cAMP Accumulation--
cAMP accumulation in
intact cells was measured according to the method of Evans et
al. (22) as described previously (16) with some modifications.
C6-2B cells on 100-mm culture dishes were incubated in F-10 medium (60 min at 37 °C) containing [2-3H]adenine (20.0 µCi/dish) to label the ATP pool. The cells were then washed once and
detached using phosphate-buffered saline-containing EDTA (0.03%). The
cells were then resuspended in a nominally Ca2+-free Krebs
buffer containing 120 mM NaCl, 4.75 mM KCl,
1.44 mM MgSO4, 11 mM glucose, 25 mM HEPES, and 0.1% bovine serum albumin (fraction V)
adjusted to pH 7.4 with 2 M Tris base. The resuspended cells were then aliquotted (approximately 3 × 105
cells/tube) and used for cAMP determination assays in triplicate. All
experiments were carried out at 30 °C in the presence of
phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine (500 µM) and Ro 20-1724 (100 µM), which were
preincubated with the cells for 10 min prior to a 1-min assay. Assays
were terminated by the addition of 10% (w/v, final concentration)
trichloroacetic acid. Unlabeled cAMP (100 µl, 10 mM), ATP
(10 µl, 65 mM), and [
-32P]ATP (~7000
cpm) were added to monitor recovery of cAMP and ATP. After pelleting,
the [3H]ATP and [3H]cAMP content of the
supernatant were quantified according to the standard Dowex/alumina
methodology (23). Accumulation of cAMP is expressed as the percentage
of conversion of [3H]ATP into [3H]cAMP;
means ± S.D. of triplicate determinations are indicated.
Electrical Recording--
Currents through CNG channels were
measured using the whole-cell patch clamp technique and an
Axopatch-200A patch clamp amplifier (Axon Instruments Inc., Foster
City, CA). Pipettes were pulled from borosilicate glass and
heat-polished. To ensure adequate voltage control in the whole cell
configuration pipette, resistance was limited to 3.5 megaohms and
averaged 2.8 ± 0.1 megaohms (n = 39). Voltage
offsets were zeroed with the pipette in the bath solution. Pipettes
were then lowered onto the cells, and gigaohms seals were formed by
applying light suction (12.8 ± 0.9 gigaohms). After achieving
whole cell configuration, capacitive transients were elicited by
applying 20-mV steps from the holding potential (0 mV), filtered at 10 kHz, and recorded at 40 kHz for calculation of access resistance and
input impedance. In all experiments, the voltage error due to series
resistance was less than 5 mV. Current records were filtered at 1 kHz,
sampled at 5 kHz, and analyzed on an IBM-compatible computer using
Pclamp6 software (Axon Instruments). The intracellular pipette filling
solution contained 145 mM KCl, 4 mM NaCl, 0.5 mM MgCl2, 10 mM HEPES, and either 0 or 1 mM cGMP, and pH was adjusted to 7.4 with KOH. The bath
solution contained 145 mM NaCl, 4 mM KCl, 10 mM HEPES, 11 mM glucose, and either 10 mM MgCl2 or 1 mM EGTA, and pH was
adjusted to 7.4 with NaOH.
[Ca2+]i
Measurements--
[Ca2+]i was measured in
populations of C6-2B cells, using fura-2 as the Ca2+
indicator, exactly as described earlier (16).
Statistics--
Analyses were performed using the PRISM
statistical software package (version 2.00, GraphPad Software,
Inc.)
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RESULTS |
Effect of Varying CNG Channel Construct Multiplicity of Infection
on Ca2+ Entry--
Initial experiments were conducted to
optimize the multiplicity of infection (m.o.i) for the adenovirus/CNG
channel construct (Ad5dl327CMV-CNGC) in C6-2B rat glioma
cells, using cytosolic Ca2+ rises
([Ca2+]i) in response to CPT-cGMP as the
functional readout (Fig. 2). Following a
48-h incubation of the cells with the CNG channel construct at an
m.o.i. of 10, 100, or 200 (A, B, or
C), the cells were loaded with fura-2 (see "Experimental
Procedures") and subsequently resuspended in a nominally
Ca2+-free Krebs buffer for Ca2+ measurements in
cell populations. Cells were pretreated with various CPT-cGMP
concentrations (0, 100, or 300 µM) 8 min prior to the
addition of [Ca2+]ex, and the resultant
[Ca2+]i rise was measured. Infection of the cells
with an m.o.i. of 10 (Fig. 2A) showed no increase in
[Ca2+]i as a result of CPT-cGMP pretreatment,
indicating little or no expression of the CNG channel. The small
[Ca2+]i rise observed following the addition of
[Ca2+]ex, from a resting level of
approximately 60 nM to approximately 180 nM
results from the cells being maintained in a nominally Ca2+-free buffer for 8 min and reflects limited CCE. When
the cells were infected at higher m.o.i. values (100 and 200; Fig. 2,
B and C), pretreatment with CPT-cGMP resulted in
a greatly augmented [Ca2+]i rise, which was
dependent on the [CPT-cGMP]. Cells infected with an m.o.i. of 100 (Fig. 2B) gave a maximal [Ca2+]i rise
to approximately 600 nM following pretreatment with 300 µM CPT-cGMP and the addition of
[Ca2+]ex. When the cells were not pretreated
with CPT-cGMP, the resultant rise in [Ca2+]i was
similar to that seen in cells infected with an m.o.i. of 10 (approximately 180 nM). Increasing the m.o.i. to 200 slightly augmented the [Ca2+]i rise, which
reached approximately 700 nM with a [CPT-cGMP] of 300 µM. Again, in the absence of CPT-cGMP pretreatment, the [Ca2+]i rise was similar to that observed in
cells not expressing the CNG channel (to approximately 170 nM, Fig. 2A). Therefore, an m.o.i of 100 or
larger results in the expression of the CNG channel. All subsequent
experiments used an m.o.i. of 100. Electrophysiological experiments
were conducted to evaluate channel expression.

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Fig. 2.
Infection of C6-2B cells with the CNG channel
construct results in functional expression of the channel. C6-2B
cells were infected with the CNG channel construct at an m.o.i. of 10 (A), 100 (B), or 200 (C) 48 h
prior to Ca2+ measurements. [Ca2+]i
was determined in aliquots of 4 × 106 fura-2-loaded
C6-2B cells as described under "Experimental Procedures." Cells in
a nominally Ca2+-free Krebs media were pretreated with
CPT-cGMP (either 0, 100, or 300 µM, as indicated) 8 min
prior to the addition of [Ca2+]ex (2 mM). Activation of the expressed CNG channel by CPT-cGMP
results in an increased [Ca2+]i rise following
the addition of [Ca2+]ex. Data are
representative of two similar experiments.
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Electrophysiological Determination of CNG Channel
Expression--
The effectiveness of using an adenovirus construct to
heterologously express the olfactory CNG channel was assessed by
monitoring currents in the whole-cell patch clamp configuration.
Currents were elicited by 250-ms steps from the holding potential, 0 mV, to membrane potentials between
80 and +60 mV in 10 mV increments, followed by a 100-ms step to
40 mV. To determine if CNG channels were
present in the infected cells, the pipette solution contained either 0 or 1 mM cGMP, which is a saturating cGMP concentration (18). The bath solution initially contained 10 mM
Mg2+, which blocks >95% of inward current and >80% of
outward current through the olfactory CNG channel (11). Thus, if CNG
channels are present and cGMP is in the patch pipette, only a small
outwardly rectifying current should be observed in the presence of 10 mM external Mg2+. Removal of Mg2+
from the bath solution would be expected to reveal a substantially larger, nonrectifying current.
When recording from cells infected with the adenovirus encoding the CNG
channel, only small leak currents (<|6 pA| at ± 40 mV) were
observed in 10 mM external Mg2+ when the patch
pipette did not contain cGMP (Fig.
3A). Removal of
Mg2+ from the bath solution caused a reversible, 2-3-fold
increase in leak (Fig. 3, B and C). The addition
of the membrane-permeant cGMP analogue, CPT-cGMP (100 µM), to the bath solution induced a large current that
was subsequently blocked by 10 mM external Mg2+
(data not shown). However, when the patch pipette contained 1 mM cGMP, a small outward current was observed in the
presence of 10 mM external Mg2+ (Fig.
3D). Removal of external Mg2+ revealed a
substantially larger, nonrectifying current that could be blocked by 10 mM Mg2+ (Fig. 3, E and
F). The collected results from 39 cells are shown in Fig.
4. Uninfected cells in the presence
(n = 9) or absence (n = 10) of cGMP and
infected cells in the absence of internal cGMP (n = 9)
displayed small inward leak currents, <15 pA at
40 mV, in 0 mM external Mg2+. Infected cells that gave a
measurable response in the presence of internal cGMP (n = 11) displayed large inward currents, >700 pA at
40 mV in 0 mM external Mg2+. The
cGMP-dependent current was observed in >70% (13 of 18, including 2 of 3 cells exposed to CPT-cGMP) of infected cells and no (0 of 9) uninfected cells.

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Fig. 3.
Electrophysiological determination of
expression of CNG channel. Currents were elicited by 250-ms steps
from the holding potential, 0 mV, to membrane potentials between 80
and +60 mV in 10 mV increments, followed by a 100-ms step to 40 mV.
A-C, currents elicited without cGMP in the pipette. Small
leak currents were observed in the presence of 10 mM
external Mg2+ (A), after washout of external
Mg2+ (B), and after the subsequent wash in of 10 mM Mg2+ (C). D-F,
currents elicited with 1 mM cGMP in the patch pipette. In
the presence of 10 mM external Mg2+,
characteristic small outward currents were observed (D).
After washout of external Mg2+, approximately equal inward
and outward currents were observed (E). These currents were
blocked by the subsequent wash in of 10 mM Mg2+
(F).
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Fig. 4.
Expression levels of CNG channels in infected
and uninfected cells. Cells infected with the CNG channel
construct displayed only small leak currents in the absence of cGMP in
the patch pipette (CNGC, 0 mM cGMP, n = 9)
in 10 mM Mg2+ (+), after the washout of
Mg2+ ( ), or after the subsequent wash in 10 mM Mg2+ (+). With cGMP in the patch pipette,
infected cells (CNGC, 1 mM cGMP, n = 11)
displayed large inward currents. Uninfected cells displayed only small
leak currents in either the absence (uninfected, 0 mM cGMP,
n = 10) or presence (uninfected, 1 mM cGMP,
n = 9) of cGMP in the pipette solution. Measurements
are presented as mean ± S.E.
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Ability of Ca2+ Entry through the CNG Channel to
Inhibit ACVI--
The next experiments aimed to determine whether the
Ca2+ entry through the expressed CNG channel could regulate
a Ca2+-inhibitable adenylyl cyclase that is endogenously
expressed in C6-2B cells. The effect of CPT-cGMP pretreatment followed
by the addition of varying [Ca2+]ex on cAMP
accumulation in uninfected versus infected cells was examined (Fig. 5). cAMP accumulation was
measured over a 1-min period following the addition of
[Ca2+]ex along with forskolin and
isoproterenol to stimulate adenylyl cyclase activity (see
"Experimental Procedures"). All cells were pretreated with the
phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (500 µM) and Ro 20-1724 (100 µM) 10 min prior to
the 1-min assay. In cells expressing the CNG channel, pretreatment with
CPT-cGMP (300 µM) caused steadily increasing inhibition
in cAMP accumulation as a function of the
[Ca2+]ex. [Ca2+]ex
of 1, 2, and 4 mM inhibited cAMP accumulation by 20, 25, and 34%, respectively. This was in contrast to uninfected cells, also pretreated with CPT-cGMP (300 µM), which gave a maximal
inhibition of 20% at a [Ca2+]ex of 4 mM. The modest degree of inhibition of cAMP accumulation seen with increasing [Ca2+]ex in the cells
not infected with the CNG channel construct was the result of limited
capacitative Ca2+ entry (see Fig. 2). The above data
support the idea that Ca2+ entry through the CNG channel
can be sensed by the Ca2+-sensitive adenylyl cyclase. To
further understand the functional relationship between the CNG channel
and the Ca2+-sensitive adenylyl cyclase, detailed
manipulations of the CPT-cGMP concentration and exposure time, as well
as the [Ca2+]ex, were carried out.

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Fig. 5.
Effects of CNGC-promoted Ca2+
entry on adenylyl cyclase type VI activity in C6-2B cells. cAMP
accumulation was measured in intact C6-2B cells as described under
"Experimental Procedures." All conditions include forskolin (10 µM) and isoproterenol (10 µM) to stimulate
adenylyl cyclase activity. Uninfected cells (circles) or CNG
channel infected cells (squares) were pretreated with
CPT-cGMP (300 µM) 10 min prior to the assay. cAMP
accumulation was measured over a 1-min period beginning with the
addition of forskolin, isoproterenol, and various
[Ca2+]ex (0, 1, 2, or 4 mM, as
indicated). Values are expressed as the percentage of cAMP accumulation
compared with the calcium-free condition (uninfected control value,
3.77; channel-infected control value, 3.18). All calcium-containing
conditions differ significantly from the relevant calcium-free
conditions, as judged by Student's t test
(p 0.0025). The uninfected data set ( CNGC) differs
significantly from the CNG channel infected data set (+CNGC), as judged
by two-way analysis of variance (p < 0.001).
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Effect of Varying [CPT-cGMP] and Exposure Time on
Ca2+ Entry and Inhibition of ACVI--
The ability of the
cGMP analog, CPT-cGMP, to activate the CNG channel partly depends on
its ability to cross the plasma membrane and reach an effective
concentration at the CNG channel. Permeation of CPT-cGMP was examined
by varying the amount of time the cells were exposed to the cGMP analog
prior to the addition of [Ca2+]ex.
Fura-2-loaded C6-2B cells were pretreated with varying amounts of
CPT-cGMP for either 2 or 5 min prior to the addition of
[Ca2+]ex. The 2-min exposure to CPT-cGMP
(Fig. 6A) resulted in a
[Ca2+]i rise following the addition of
[Ca2+]ex that depended on the [CPT-cGMP].
At the highest [CPT-cGMP], 100 µM, the
[Ca2+]i rise reached a peak of approximately 370 nM after approximately 2 min. The 50 µM
CPT-cGMP condition did not reach a plateau in the course of the
experiment but reached a slightly lower maximum value (approximately
330 nM) than the 100 µM condition. At even
lower [CPT-cGMP], 20 µM, the rate of the
[Ca2+]i rise was even slower, and the maximum
value achieved was substantially less (approximately 200 nM). In the absence of CPT-cGMP pretreatment, a very modest
[Ca2+]i rise was observed, due to the fact that
the cells were being maintained in a nominally Ca2+-free
medium. When the time of exposure to CPT-cGMP was increased to 5 min,
the rate of the [Ca2+]i rise following the
addition of [Ca2+]ex was considerably faster,
reaching a plateau within the course of the experiment (Fig.
6B). Although the rates of the [Ca2+]i
rise increased with longer exposure to CPT-cGMP, the peak values
reached were very similar to the 2-min exposure (see Fig.
6A) with 20, 50, and 100 µM CPT-cGMP
treatments reaching peaks of approximately 200, 320, and 360 nM, respectively. These results showed that permeation of
the cGMP analog across the plasma membrane is rather slow, but once
maximal activation of the channel has been reached, the peak
[Ca2+]i rises are very similar for a given
[CPT-cGMP].

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Fig. 6.
Effect of varying [CPT-cGMP] and exposure
time on Ca2+ entry in C6-2B cells expressing the CNG
channel. [Ca2+]i was measured in cells
infected with the CNG channel construct in nominally
Ca2+-free Krebs buffer as described under "Experimental
Procedures." Cells were pretreated with various CPT-cGMP
concentrations (0, 20, 50, or 100 µM, as indicated)
either 2 min (A) or 5 min (B) prior to the
addition of [Ca2+]ex (2 mM).
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In order to consolidate the regulatory consequence of Ca2+
entry through the CNG channel on the adenylyl cyclase, different CPT-cGMP exposure times were compared in terms of their effect on cAMP
accumulation and Ca2+ entry. Fig.
7 shows the effect of varying both the
CPT-cGMP concentration and the exposure time to the cGMP analog on the
cAMP accumulation in C6-2B cells infected with the CNG channel
construct. Following a 2-min exposure to CPT-cGMP, a combination of
[Ca2+]ex as well as forskolin and
isoproterenol were added to the cells with cAMP accumulation measured
over the subsequent minute. With increasing [CPT-cGMP], there was a
stepwise increase in the inhibition of cAMP accumulation ranging from
7% inhibition with 20 µM CPT-cGMP to 32% with 300 µM CPT-cGMP (Fig. 7A). It should be noted that the extent of inhibition observed with increasing [CPT-cGMP] agrees well with the extent of Ca2+ entry (Fig. 6A).
Without CPT-cGMP pretreatment of the cells, a minimal inhibition of
cAMP accumulation (8%) was observed, which is very similar to the 20 µM CPT-cGMP condition. As seen in Fig. 6A, 0 and 20 µM CPT-cGMP produce a similar
[Ca2+]i rise within the first minute, the period
over which cAMP accumulation is measured. Therefore, the similarities
in the extent of the inhibition seen with 0 and 20 µM
CPT-cGMP are consistent with the Ca2+ data. In Fig.
7B, the effects of a 5-min exposure to varying CPT-cGMP
concentrations on cAMP accumulation are shown. Again, increasing the
CPT-cGMP concentration produced further inhibition of cAMP accumulation
following the addition of [Ca2+]ex, with
maximal inhibition (31%) observed at 300 µM CPT-cGMP. The amount of inhibition observed in the absence of CPT-cGMP was again
8%, which, following a 5-min exposure to CPT-cGMP, differs greatly
from the 20 µM CPT-cGMP condition (22%). This result was also in good agreement with the corresponding Ca2+ data
(Fig. 6B), where the longer pretreatment with CPT-cGMP
resulted in a faster [Ca2+]i rise and, therefore,
a higher [Ca2+]i level achieved within the 1-min
assay period. It is also noteworthy that the extent of inhibition in
cAMP accumulation appears to reach "maximal" levels at lower
CPT-cGMP concentrations with these longer exposure times. In other
words, the dose-response curve has been shifted to the left, indicating
an increased efficacy in the Ca2+ entry promoted by
increasing CPT-cGMP concentrations to inhibit the cyclase. This agrees
well with the Ca2+ data, which showed that increasing the
exposure time to CPT-cGMP prior to the addition of
[Ca2+]ex produced a more rapid
[Ca2+]i rise that reaches a plateau more
rapidly.

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Fig. 7.
Effect of varying [CPT-cGMP] and exposure
time on the ability of CNGC-promoted Ca2+ entry to regulate
cAMP accumulation. Cells infected with the CNG channel construct
were pretreated with varying CPT-cGMP concentrations (0, 20, 50, or 100 µM, as indicated) in a nominally Ca2+-free
Krebs buffer either 2 min (A) or 5 min (B) prior
to cAMP determination. cAMP accumulation was measured over a 1-min
period in the presence of forskolin (10 µM),
isoproterenol (10 µM), and added
[Ca2+]ex (either 0 or 2 mM). The
asterisks denote values that differ significantly from the
relevant control (0 CPT-cGMP/2 mM Ca2+
condition), as judged by Student's t test
(p < 0.01).
|
|
Effect of Varying [CPT-cGMP] and
[Ca2+]ex on Ca2+ Entry and
Inhibition of ACVI--
The next set of experiments was designed to
examine the effect of varying both the
[Ca2+]ex and the [CPT-cGMP] on the
resultant [Ca2+]i rise and inhibition of cAMP
accumulation in C6-2B cells infected with the CNG channel construct.
Fura-2-loaded cells were pretreated with 0, 50, or 100 µM
CPT-cGMP 4 min prior to the addition of 1, 2, or 4 mM
[Ca2+]ex, and the ensuing
[Ca2+]i rise was measured (Fig.
8). The addition of 1 mM
[Ca2+]ex yielded a
[Ca2+]i rise of approximately 80, 170, and 210 nM following treatment of the cells with 0, 50, or 100 µM CPT-cGMP, respectively (Fig. 8A).
Increasing the [Ca2+]ex to 2 mM
produced a more pronounced [Ca2+]i rise, which
reached approximately 130, 300, and 350 nM with 0, 50, and
100 µM CPT-cGMP treatment, respectively (Fig. 8B). A further increase in
[Ca2+]ex to 4 mM produced still
larger [Ca2+]i rises ranging from approximately
220 to 430 nM with increases in CPT-cGMP concentrations
from 0 to 100 µM. Therefore, at any CPT-cGMP
concentration used, raising [Ca2+]ex produced
corresponding increases in [Ca2+]i.

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Fig. 8.
Effect of varying [CPT-cGMP] and
[Ca2+]ex on Ca2+ entry in C6-2B
cells expressing the CNG channel. [Ca2+]i
was measured in aliquots of C6-2B cells infected with the CNG channel
construct in nominally Ca2+-free Krebs buffer as described
under "Experimental Procedures." Cells were pretreated with various
CPT-cGMP concentrations (0, 50, or 100 µM, as indicated)
4 min prior to the addition of [Ca2+]ex. The
resultant rise in [Ca2+]i is shown following the
addition of 1 mM [Ca2+]ex
(A), 2 mM [Ca2+]ex
(B), or 4 mM [Ca2+]ex
(C).
|
|
Next, the effect on cAMP accumulation of the incremental increases in
[Ca2+]i caused by increasing
[Ca2+]ex following CPT-cGMP treatment was
examined. Fig. 9 depicts the effect of
Ca2+ entry through CNG channels promoted by treatment with
either 50 or 100 µM CPT-cGMP on cAMP accumulation in
C6-2B cells infected with the CNG channel construct. Increasing the
[Ca2+]ex from 0 to 4 mM resulted
in an increased inhibition in cAMP accumulation with both [CPT-cGMP].
The cells pretreated with 100 µM CPT-cGMP yielded the
largest inhibition, maximally 32% (Fig. 9). Once again, experimental
conditions that alter the [Ca2+]i rise produced
by Ca2+ entry through the CNG channel achieve corresponding
changes in the inhibition of cAMP accumulation. The data above clearly
show that Ca2+ entry through an expressed CNG channel
regulates the endogenously expressed Ca2+-inhibitable
adenylyl cyclase. Finally, it was of interest to compare regulation of
cAMP accumulation by the CNG channel with the normal physiological mode
of Ca2+ regulation of cAMP accumulation in these cells,
i.e. capacitative Ca2+ entry.

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Fig. 9.
Effect of varying [CPT-cGMP] and
[Ca2+]ex on the ability of CNGC-promoted
Ca2+ entry to regulate cAMP accumulation. Cells
expressing the CNG channel were pretreated with CPT-cGMP (50 µM, open bars; 100 µM,
hatched bars) in nominally Ca2+-free Krebs
buffer 4 min prior to cAMP determination. cAMP accumulation was
measured over a 1-min period in the presence of forskolin (10 µM), isoproterenol (10 µM), and added
[Ca2+]ex (0, 1, 2, or 4 mM, as
indicated). The asterisks denote values that differ
significantly from the relevant controls, as judged by Student's
t test (p < 0.005).
|
|
Comparison of the Efficacy of CNG Channel-promoted Ca2+
Entry Versus Capacitative Ca2+ Entry--
We had
previously established the exclusive ability of capacitative
Ca2+ entry to regulate the endogenously expressed
Ca2+-inhibitable adenylyl cyclase in C6-2B cells (16).
Other modes of inducing [Ca2+]i rises, such as
release from intracellular stores or an extremely robust
[Ca2+]i rise produced by ionophore treatment,
were ineffective (16). Therefore, the ability of a
[Ca2+]i rise emanating from expressed CNG
channels to regulate the cyclase was somewhat unexpected. The next set
of experiments was designed to examine the relative efficacy of these
two forms of Ca2+ entry (endogenous CCE versus
heterologously expressed, CNG channel-promoted Ca2+ entry)
to regulate cAMP accumulation in C6-2B cells. Fig.
10A depicts the
[Ca2+]i rise in cells infected with the CNG
channel construct treated with CPT-cGMP (100 µM) 4 min
prior to the addition of varying [Ca2+]ex.
The addition of 1, 2, or 4 mM
[Ca2+]ex produced peak
[Ca2+]i rises of approximately 220, 280, or 330 nM within 1 min. These [Ca2+]i rises
were modest in comparison with CCE (cf. Fig. 10B). CCE was promoted by treating the cells with
thapsigargin (TG; 100 nM), which inhibits the
Ca2+-ATPase responsible for pumping Ca2+ into
the stores (24), leaving the endogenous Ca2+ leak to
deplete the stores. The depletion of the intracellular Ca2+
stores promotes the subsequent CCE. Following depletion of
intracellular Ca2+ stores, varying
[Ca2+]ex in the media resulted in a rapid
rise in [Ca2+]i. The addition of 1 mM
[Ca2+]ex to the TG-treated cells resulted in
a [Ca2+]i rise to approximately 520 nM, while the addition of 2 and 4 mM evoked
peak [Ca2+]i rises of approximately 680 and 860 nM, respectively (Fig. 10B). Next, the effects
of these two modes of Ca2+ entry were compared with respect
to their ability to regulate cAMP accumulation (Fig.
11). In both Ca2+ entry
protocols, an increase in the amount of inhibition in cAMP accumulation
was observed with increasing [Ca2+]ex. CCE
produced greater inhibition of the cyclase, with a maximal inhibition
of 40%, using a [Ca2+]ex of 4 mM. In comparison, CNG channel-promoted Ca2+
entry inhibited cAMP accumulation by 32% at the same
[Ca2+]ex. At first glance, it may appear that
CCE is more effective at regulating cAMP accumulation, but when the
[Ca2+]i rise produced by these two
Ca2+ entry methods is compared (see Fig. 10), it may be
argued that qualitatively they are very similar in their efficacy. CNG
channel-promoted Ca2+ entry gave a maximal
[Ca2+]i rise of approximately 330 nM
with a [Ca2+]ex of 4 mM, and CCE
produced a [Ca2+]i rise of approximately 860 nM under the same conditions (Fig. 10). Therefore, there is
a rough correlation between the [Ca2+]i levels
reached and the amount of inhibition of cAMP accumulation observed.
This finding argues that both of these Ca2+ entry methods
are equally efficacious in regulating adenylyl cyclase activity.

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Fig. 10.
Comparison of CNGC-promoted and capacitative
Ca2+ entry in C6-2B cells.
[Ca2+]i was measured in cells expressing the CNG
channel construct in nominally Ca2+-free Krebs buffer as
described under "Experimental Procedures." In A,
CNGC-promoted Ca2+ entry was stimulated in C6-2B cells
expressing the CNG channel by pretreatment with CPT-cGMP (100 µM) 4 min prior to the addition of
[Ca2+]ex (1, 2, or 4 mM, as
indicated). Capacitative Ca2+ entry is depicted in
B, where C6-2B cells were pretreated with TG (100 nM), which produces a [Ca2+]i rise
due to Ca2+ release from intracellular stores. Following
depletion of Ca2+ stores,
[Ca2+]ex (1, 2, or 4 mM, as
indicated) was added to the media, with the resultant capacitative
Ca2+ entry depicted.
|
|

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Fig. 11.
Comparison of the efficacy of CNGC-promoted
versus capacitative Ca2+ entry in
regulating cAMP accumulation. Cells expressing the CNG channel
were pretreated with CPT-cGMP (100 µM,
circles) or TG (100 nM, triangles) in
nominally Ca2+-free Krebs buffer 4 min prior to cAMP
determination. cAMP accumulation was measured over a 1-min period in
the presence of forskolin (10 µM), isoproterenol (10 µM), and added [Ca2+]ex (0, 1, 2, or 4 mM, as indicated). Values are expressed as the
percentage of cAMP accumulation compared with the calcium-free
condition (channel-infected control, 2.66; TG-treated control, 2.47).
All calcium-containing conditions differ significantly from the
relevant calcium-free conditions, as judged by Student's t
test (p < 0.005). The CPT-cGMP-treated data set
does not differ significantly from the TG-treated data set,
as judged by two-way analysis of variance.
|
|
 |
DISCUSSION |
The present study has established that Ca2+ entry
through a heterologously expressed CNG channel can regulate the
endogenous Ca2+-inhibitable adenylyl cyclase of C6-2B
cells. Electrophysiological measurements showed that infection using
the novel adenovirus construct coding for the olfactory CNG channel
-subunit achieved expression in more than 70% of the cells, which
is remarkable, given the refractory nature of these cells to transient
transfection. The expressed channel behaved normally, based on cyclic
nucleotide dependence, conductance, and Mg2+ block.
Subsequently, substantial Ca2+ entry was observed in
populations of cells, which was dependent not only on the [CPT-cGMP]
and [Ca2+]ex but also on the time of exposure
to CPT-cGMP. The Ca2+ entry through the CNG channel
inhibited the endogenous adenylyl cyclase activity of C6-2B cells. The
degree of inhibition mirrored the magnitude of the
[Ca2+]i rise generated by the various
experimental conditions. For instance, a relatively small
[Ca2+]i rise generated either by low [CPT-cGMP]
or short pretreatment times caused a relatively small inhibition of the
adenylyl cyclase. When the [CPT-cGMP] or exposure time was increased,
the degree of inhibition of the cyclase was increased.
We had previously shown that capacitative Ca2+ entry
regulates the Ca2+-sensitive adenylyl cyclase in
nonexcitable cells, whether the cyclase was endogenously or
heterologously expressed (15, 16, 25). Even extremely high
[Ca2+]i levels, achieved as a consequence of
ionophore treatment, were unable to regulate the adenylyl cyclase
activity. These and other data (15, 16, 25) led us to suggest that the
adenylyl cyclase and Ca2+ entry channels must be located in
similar microdomains in the cell. Therefore, the present findings, that
Ca2+ entry through a heterologously expressed CNG channel
regulates adenylyl cyclase, were somewhat unexpected. Indeed, when CCE
and CNG channel-promoted Ca2+ entry were compared, it was
quite evident that they were equally efficacious at modulating adenylyl
cyclase activity. Although CCE could achieve slightly greater
inhibition of cAMP accumulation compared with CNG channel-promoted
Ca2+ entry (40 versus 32%, respectively), the
[Ca2+]i rise produced by CCE was substantially
larger at a given [Ca2+]ex. Extending the
rationale that led us to conclude that the CCE channel is functionally
colocalized with the Ca2+-sensitive adenylyl cyclase, it
can also be asserted that the CNG channel must also allow
Ca2+ entry in the vicinity of the cyclase and, therefore,
be targeted to this same domain.
The present findings not only point to a functional colocalization
between CNG channels and the Ca2+-sensitive adenylyl
cyclase, they also strengthen the notion that CNG channels may function
as a pathway for Ca2+ entry that is not dependent on
Ca2+ store depletion or membrane depolarization. It has
been clear for some time that Ca2+ entry through CNG
channels plays an important role in transduction and adaptation in
visual and olfactory receptors (Refs. 26-28; reviewed in Refs. 29 and
30). In the cone synapse, it has been shown that CNG channels, as well
as voltage-gated Ca2+ channels, are involved in
exocytosis of synaptic vesicles (31). Furthermore, it has been
shown that exocytosis in cone synapses can be modulated by NO, by
affecting cGMP production and altering CNG channel activity (32).
CNG channels have also been postulated to play a role in synaptic
plasticity, a process that is dependent on Ca2+. In the
hippocampus, an olfactory-like CNG channel has been found in cell
bodies and processes of CA1 and CA3 neurons (8), which express high
levels of two Ca2+-stimulable adenylyl cyclases, types I
and VIII (33). Based on these observations, it has been suggested that
modulation of adenylyl cyclase activity by Ca2+ entry
through the CNG channel in CA1 neurons may participate in maintenance
of long term potentiation (8). Evidence in support of this proposal is
that hippocampi isolated from an olfactory CNG channel null mouse were
impaired in their ability to produce long term potentiation in response
to
-burst stimulation (34). Another tissue in which CNG channels
have been detected is the heart (4, 9). The heart is also one of the
most abundant sources of Ca2+-inhibitable adenylyl
cyclases, types V and VI (35, 36). We had earlier proposed that the
existence of feedback loops between cAMP-controlled Ca2+
entry and Ca2+-inhibitable adenylyl cyclases could give
rise to oscillations in both [cAMP] and [Ca2+]i
(14). The present finding that Ca2+ entry through a CNG
channel can inhibit a Ca2+-inhibitable adenylyl cyclase may
provide a molecular basis for such a proposal.
For the present, the ability of Ca2+ entry through a
heterologously expressed CNG channel to regulate a
Ca2+-sensitive adenylyl cyclase extends earlier
observations that endogenous CCE mechanisms could regulate
heterologously expressed adenylyl cyclases (15). This finding may
suggest that Ca2+-sensitive adenylyl cyclases and
Ca2+ entry mechanisms are endowed with common
characteristics, such as preferential solubility in cholesterol-rich
domains (37), that ensure their coincidence in microdomains of the
plasma membrane.
 |
ACKNOWLEDGEMENTS |
We thank Elizabeth Ullyat for excellent
technical support and Robert Graf for helpful discussions. We also
thank Dr. R. R. Reed for providing the cDNA encoding the rat
olfactory cyclic nucleotide-gated channel used in this study.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
(NIH) Grants GM 32483 and NS 28389 (to D. M. F. C.) and HL58344 (to J. S.) and NCI, NIH, Grant P30-CA46934 (to the Cancer Center of the
University of Colorado).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Pharmacology, Box C-236, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-8964; Fax: 303-315-7097; E-mail: cooperd{at}essex.uchsc.edu.
2
J. Schaack, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
CNG channel, cyclic
nucleotide-gated channel;
CCE, capacitative calcium entry;
[Ca2+]i, cytosolic Ca2+
concentration;
[Ca2+]ex, extracellular
Ca2+ concentration;
CPT-cGMP, 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate;
m.o.i., multiplicity of infection;
TG, thapsigargin;
ACVI, adenylyl cyclase
type VI;
CNGC, CNG channel;
TP, terminal protein.
 |
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