Adenovirus-mediated Expression of an Olfactory Cyclic Nucleotide-gated Channel Regulates the Endogenous Ca2+-inhibitable Adenylyl Cyclase in C6-2B Glioma Cells*

Kent A. FaganDagger , Thomas C. Rich§, Shawna Tolman, Jerome Schaack, Jeffrey W. Karpen§, and Dermot M. F. CooperDagger parallel

From the Departments of Dagger  Pharmacology, § Physiology and Biophysics, and  Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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
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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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Materials-- Thapsigargin, forskolin, and Ro 20-1724 were from Calbiochem. [2-3H]Adenine and [alpha -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 alpha -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 Ad5dl327Bstbeta -Gal-TP complex (20). Ad5dl327Bstbeta -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 beta -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 Ad5dl327Bstbeta -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 Ad5dl327Bstbeta -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-beta -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 beta -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.

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 [alpha -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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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.

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.

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).

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).

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 theta -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.

parallel 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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