Correspondence to: Jeffrey W. Karpen, Dept. of Physiology and Biophysics, Box C-240, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. Fax:303-315-8110 E-mail:jeffrey.karpen{at}uchsc.edu.
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Abstract |
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Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca2+, cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotidegated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotidegated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 2256 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotidegated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotidegated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.
Key Words: intracellular signaling, cyclic AMP, calcium influx, microdomains, subcellular compartments
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INTRODUCTION |
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The second messenger cyclic AMP (cAMP) regulates a large variety of cellular processes including Ca2+ influx (
The endogenous cyclic nucleotidegated channels in retinal rods have been used as a high-resolution monitor of cGMP signaling. These measurements have led to quantitative descriptions of the single photon response (
Studies using endogenous cyclic nucleotidegated channels have been limited to only a few other cell types, including olfactory receptors (
Recently, cyclic nucleotidegated channels have been artificially introduced into other cell types using the "patch-cramming" technique (
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MATERIALS AND METHODS |
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Cell Culture and Expression
Both human embryonic kidney 293 (HEK-293) cells and HEK-293 cells stably expressing type 8 AC (HEK-AC8) were maintained in 10 ml of MEM (Life Technologies) with 10% vol/vol fetal bovine serum (Gemini) and 0.1 mg/ml G418, and grown in 75 cm2 flasks at 37°C in a humidified atmosphere of 95% air/5% CO2. Cells were plated at ~60% confluence in 100-mm culture dishes for infection with the oCNG channel-encoding adenovirus construct (
Electrical Recording
Recordings were made at room temperature (1921°C) using an Axopatch-200A patch-clamp amplifier (Axon Instruments, Inc.) and whole-cell, perforated patch, and excised patch techniques. Pipettes were pulled from borosilicate glass and heat polished. Pipettes were lowered onto the cells and gigaohm seals were formed (8.3 ± 3.3 G). Pipette resistance was limited to 5 M
and averaged 3.4 ± 1.0 M
. Current records were typically sampled at five times the filter setting and stored on an IBM compatible computer. Records were corrected for errors due to series resistance. Unless otherwise noted, all data are presented as mean ± SD. The solution within the chamber (100 µl) was changed within 15 s using a gravity-driven perfusion system.
Whole-cell and perforated patch configurations.
After achieving whole-cell or perforated patch configuration, capacitive transients were elicited by applying 20-mV steps from the holding potential and recorded at 40 kHz (filtered at 10 kHz) for calculation of access resistance. In the whole-cell configuration, the access resistance averaged 6.6 ± 3.1 M. No significant difference in access resistance was observed in whole-cell experiments performed under different experimental conditions. Access resistance was monitored throughout the experiments to ensure stable electrical access was maintained. The solutions used for all of the experiments are listed in Table 1. Whole-cell pipette solution 1 (Table 1) was used as the control solution for all whole-cell experiments. In the perforated patch configuration, pipette solutions (perforated patch 1 and 2, Table 1) contained nystatin (diluted from 50 mg/ml stock in dimethylsulfoxide) to gain electrical access to the cell. These solutions were kept on ice and shielded from light until use. A steady access resistance (1560 M
) was obtained 515 min after seal formation. Nystatin did not induce measurable currents up to 20 min after break-in in whole-cell experiments. Voltage protocols were selected to minimize endogenous currents.
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Excised, inside-out patch configuration. Excised patch solution (Table 1) was used in both the pipette and bath. Cyclic AMP was added to the bath solution in concentrations between 1 and 1,000 µM. cAMP-induced currents were obtained from the difference between currents in the presence and absence of cAMP.
Photolysis of NPE-cAMP
The response of oCNG channels to flash photolysis of NPE-cAMP was measured in the whole-cell patch-clamp configuration. The pipette filling solution (whole-cell pipette 2 or 3, Table 1) was kept on ice and shielded from light until use. After achieving whole-cell configuration, we waited 10 min before photolysis of NPE-cAMP. Photolysis of NPE-cAMP was achieved by direct flash (1 ms) of the entire bath with an XF-10 xenon flashlamp (HI-TECH Scientific). The DMSO used to dissolve NPE-cAMP and nystatin (0.5% final concentration) had no effect on channels in excised patches.
Determination of Total cAMP in C6-2B Cells
C6-2B cells were suspended in low-Mg bath 1 solution (Table 1) for 10 min at 20°C. cAMP accumulation was initiated by addition of forskolin or vehicle for 2.5 min and terminated by addition of trichloroacetic acid (9% wt/vol). The total intracellular levels of cAMP were measured by a cAMP binding assay (Amersham Pharmacia Biotech) as previously described (
Measurement of Ca2+
Intracellular [Ca2+] was measured in cell populations using fura-2 as the Ca2+ indicator, as previously described (
Forskolin was from Calbiochem. Fura-2/AM, Pluronic F-127, and NPE-cAMP were from Molecular Probes. All other chemicals were from Sigma-Aldrich.
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RESULTS |
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Cyclic nucleotidegated channels have the following properties that led us to investigate their utility as cAMP sensors. First, oCNG channels can be expressed easily in a variety of cell types using a recently described adenovirus construct encoding the subunit (
Response of oCNG Channels to Jumps in cAMP
The response times of homomultimeric oCNG channels to changes in cAMP have not been established. We examined the ionic currents induced by perturbations in cellular cAMP levels generated by photolysis of a "caged" cAMP analogue, NPE-cAMP. oCNG channels were expressed in the HEK-AC8 cell line and currents were monitored using the whole-cell patch-clamp technique. The pipette solution (whole-cell pipette 2, Table 1) contained 200 µM NPE-cAMP and 20 µM cAMP to verify that there was channel activity, and to prime the channels to give a larger flash response. The bath solution initially contained 0.1 mM MgCl2 (low-Mg bath 1, Table 1) to allow measurement of inward current through oCNG channels at a membrane potential of -50 mV. After a 1-ms flash, there was a brief delay, 14.8 ± 4.1 ms (n = 4), followed by an exponential increase in current, = 212 ± 53 ms (Fig 1 A). This current was subsequently blocked by 10 mM MgCl2 (high-Mg bath 1, Table 1) and, in the continued presence of 10 mM MgCl2, repeated photolysis of NPE-cAMP did not induce a detectable inward current (Fig 1 A). When the external MgCl2 concentration was returned to 0.1 mM, current through oCNG channels was no longer blocked and could again be induced by photolysis of NPE-cAMP. No currents were induced by the photolysis of NPE-cAMP when 1 mM cAMP (a supersaturating concentration) was added to the pipette solution (whole-cell pipette 3, Table 1) or in control cells not infected with the oCNG-encoding adenovirus construct (Fig 1 B). The kinetics of the cAMP-induced current were similar to the photolysis rate of NPE-cAMP (~4 s-1 at pH = 7.4), indicating that the photolysis rate of NPE-cAMP was the rate-limiting step in the response (
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Calibration of the Sensor
We determined the apparent cAMP affinity of oCNG channels in excised, inside-out patches from HEK-AC8 cells. After patch excision, currents were elicited by applying solutions containing between 0 and 1,000 µM cAMP at membrane potentials (Vm) of +50 and -50 mV. In excised patches expressing oCNG channels, increasing cAMP concentration induced larger currents until a saturating cAMP concentration was reached (Fig 2 A). No cAMP-inducible current was observed in control cells. The sensitivity of oCNG channels to cAMP (Fig 2 B) was quantified using the Hill equation (Fig 2, legend), yielding an average apparent affinity (K1/2) and Hill coefficient (N) of 40 ± 13 µM and 2.1 ± 0.2 (measured at -50 mV, n = 23), similar to previously reported values for channels expressed in HEK-293 cells (
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Single Cell Measurement of cAMP Using oCNG Channels
To determine whether heterologously expressed oCNG channels could detect cAMP in single cells, we chose to first examine cAMP accumulation in the HEK-AC8 cell line. Adenylyl cyclase activity was stimulated with 50 µM forskolin2 (added to low-Mg bath 1 solution, Table 1) and the subsequent activation of oCNG channels was monitored using the perforated patch technique (
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To ensure that the high cAMP concentration measured by the channel was not due to the heterologous expression of AC in the HEK-AC8 cell line, we also examined the generation of cAMP by the endogenous type 6 AC in C6-2B glioma cells (
This concentration is far higher than the low micromolar levels of cAMP measured under the same stimulus conditions in single C6-2B cells using fluorescently labeled PKA subunits (
cAMP Accumulates Despite Rapid Dialysis of the Bulk Cytosol
If cAMP is generated in diffusionally limited microdomains, then forskolin-induced cAMP accumulation should be detectable in the whole-cell configuration in which there is rapid exchange of the bulk cytoplasm (with whole-cell pipette solution 4, Table 1). We tested this prediction using HEK-AC8 cells expressing oCNG channels. After stimulation of AC with 50 µM forskolin, a steady rise to substantial current levels was observed in five of nine cells (Fig 4 A), consistent with expression in 70% of cells (56 s (see below), yet the forskolin-induced currents increased for several minutes at rates similar to those observed in cells studied in the nondialyzed perforated patch configuration. This finding strongly argues that cAMP diffusion from microdomains into the bulk cytosol is impeded. In the next two sections, we show quantitatively that (a) the results cannot be explained if cAMP diffuses freely throughout the cell, and (b) the results can be explained by cell compartment models in which there is a barrier to cAMP diffusion between the microdomains and the bulk cytosol. These models are supported further by experiments in which the flux of cAMP is measured from the whole-cell pipette to the channels.
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Free Diffusion of cAMP from Adenylyl Cyclase
In this section, we show that, with no diffusion restriction, cAMP concentrations right next to AC are not high enough to substantially activate PKA, let alone oCNG channels. Let us consider the local concentration of cAMP near AC. The diffusion equation for constant diffusion coefficient D is written as (
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(1) |
where C is the concentration of cAMP. If we approximate the AC molecule as a point source on an impermeable plane, Equation 1 can be solved analytically, yielding (
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(2) |
where kcat is the turnover number, x is the distance from the catalytic site of AC, and t is time. Given that the radius of HEK-AC8 cells is ~10 µm and that we are estimating cAMP concentrations 10 nm (within molecular dimensions) from AC, this geometric approximation is valid. For the simulation, we assumed a diffusion coefficient for cAMP in cytoplasm of 3 x 10-6 cm2/s (
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It is useful to compare this low cAMP concentration with the high concentration of Ca2+ reached near the mouth of a Ca2+ channel (Fig 5 B, see legend for details). The high steady state Ca2+ concentration near the channel (88 µM at 5 nm) is similar to previous estimates of local Ca2+ concentration (
With no restriction on diffusion, the steady state cAMP concentration near AC is reached rapidly (within 2 ms, Fig 5 C). This is much faster than the time course we observed (~150 s, see Fig 3). The solution in the experimental chamber was exchanged in ~15 s and forskolin activation of AC occurs within 8 s (
Cell Compartment Models
Fig 6 shows a simple compartment model of the cell and whole-cell patch pipette. The cell contains two compartments: a microdomain (compartment 1) in which AC produces cAMP, and the bulk cytosol (compartment 2). The pipette is represented by compartment 3. In simulating the flux of cAMP between the compartments, we have made the following assumptions: (a) diffusion between compartments is restricted by a barrier, and (b) diffusion within each compartment is much more rapid than diffusion between compartments. Thus, the concentration in each compartment is considered to be uniform (rapidly equilibrated) at all times. The second assumption is justified, based on how rapidly cAMP would diffuse without restriction across the entire cell. Using the equation for diffusion in three dimensions: x 2
= 6Dt, cAMP can diffuse across a C6-2B cell (x = 16.8 µm) in 160 ms. This is considerably faster than the exchange times between cellular compartments (see below).
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This system is described by the following three equations (Equation 3 HREF="#FD4">Equation 4Equation 5):
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(3) |
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(4) |
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(5) |
where V1, V2, and V3 are the volumes of each compartment, C1, C2, and C3 are the concentrations in each compartment, and J12 and J23 are flux coefficients between compartments 1 and 2, and compartments 2 and 3, respectively.
Before using this model to simulate the accumulation of cAMP synthesized by AC, we sought to experimentally determine the rates of transfer of material between the compartments in HEK-293 cells. To estimate the rate of transfer between the pipette and the bulk cytosol (compartments 3 and 2), we first measured the reduction in endogenous, voltage-gated K+ currents in response to the wash-in of a solution containing high NaCl and low KCl (whole-cell pipette 5, Table 1). After break-in, currents were measured every 2 s in response to 40-ms pulses to +60 mV from a holding potential of -80 mV (Fig 7). This protocol allowed us to monitor the endogenous K+ currents without rundown. When the pipette solution contained high NaCl, the amplitude of the outward current decreased rapidly (90% in 22 ± 4 s, n = 4). To ensure that the results were not affected significantly by Na+ block of the K+ currents, we also examined tail currents at -40 mV and obtained a similar time course. No reduction of current was observed when the pipette solution contained high KCl (whole-cell pipette 1, Table 1). To estimate the rate of transfer of cAMP based on these results, we scaled the wash-in time by the ratio of diffusion coefficients for Na+ and cAMP (3.5;
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To estimate the rate of transfer of cAMP between the cytosol and the microdomain (compartments 2 and 1), we measured the development of currents through oCNG channels in response to the wash-in of 40 µM or 1 mM cAMP from the patch pipette. These experiments were done with high concentrations of two PDE inhibitors in both the bath (low-Mg bath 2, Table 1) and the pipette (whole-cell pipette 6 or 7) solutions. The cells were exposed to the bath solution containing PDE inhibitors for >6 min before break in. As before, currents through oCNG channels were verified by Mg2+ block (high-Mg bath 2 solution, Table 1). Fig 8 A shows that, after break in with 40 µM cAMP (a half-saturating concentration) in the pipette, a current developed slowly (90% complete in 194 ± 62 s, n = 5). This time course was considerably slower than the exchange of the bulk cytosol, and thus represents another clear demonstration of the diffusional barrier between the cytosol and the microdomain. The time course with 1 mM cAMP in the pipette was much faster (90% complete in 38 ± 16 s, n = 7), consistent with this being a supersaturating concentration and high concentrations reaching the channel well before exchange was complete (Fig 8 B). This result also indicates that cAMP buffering is not the primary restriction on diffusion.3
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Interestingly, when 0.5 µM pCPT-cGMP (8-p-chlorophenylthio-cGMP) was included in the pipette solution (whole-cell pipette 8, Table 1), the 90% response time of oCNG channels was 38 ± 18 s, n = 10 (Fig 8 C). This concentration of pCPT-cGMP is 4.5x greater than K1/2 (~110 nM), and thus the 90% response time we measured was faster than would be expected at lower pCPT-cGMP concentrations. When this was taken into account, the 90% response time was calculated to be 56 s. The membrane-permeant pCPT-cGMP was apparently able to traverse the barrier between the cytosol and the microdomain more rapidly than cAMP (56 vs. 194 s). Thus, the 56-s wash-in time of pCPT-cGMP can be considered an upper limit for the 90% exchange time between the whole-cell pipette and the bulk cytosol, and was used to constrain J 23. The wash-in times for Na+ and pCPT-cGMP are consistent with those measured in a previous study of chromaffin cells (
We now show that the cell compartment model in Fig 6 can describe the transfer of cAMP between compartments, the high local and low cytosolic cAMP following AC activation, and the accumulation of cAMP despite rapid dialysis of the cytosol. We used a cytosolic volume of 2.5 pl (based on a cell radius of 8.4 µm), and assumed the volume of the microdomain is 0.04 pl (<2% of the cell volume). Values for the flux coefficients J12 and J 23 of 8.0 x 10-16 liters/s and 9.0 x 10-14 liters/s allowed us to describe the exchange of the bulk cytosol and the wash-in of 40 µM cAMP (for the latter, compare Fig 9 A and 8 A). The 40 µM cAMP time course is dominated by the slow exchange between the cytosol and microdomain, but the finite exchange time between the pipette and cytosol, and the nonlinear response of oCNG channels to cAMP, contribute to the sigmoidal shape. Using the same parameters, we are able to describe the wash-in of 1 mM cAMP (Fig 9 B).3 Assuming that there are 450 ACs (each with a kcat of 59 s-1), we can also account for the buildup of high concentrations of cAMP in the microdomain (Fig 9 C), the accumulation of low concentrations in the bulk cytosol (Fig 9 C), and the negligible effect of rapid dialysis of the cytosol on the cAMP buildup within the microdomain (Fig 9 D). We have not attempted to model the lag that is due to (a) the perfusion time (~15 s), (b) the time it takes forskolin to cross the membrane (~8 s;
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Assuming that the flux can be described by simple diffusion, the flux coefficient is related to the classically defined quantities permeability (p) and diffusion coefficient (D) by the relations J = P · A = D · A/l , where A is the cross-sectional area and l is the thickness of the barrier. The flux coefficient between the whole-cell pipette and the cytosol (J 23 = 9.0 x 10-14 liters/s) can be explained by a diffusion coefficient of 3 x 10-6 cm2/s (the cytosolic diffusion coefficient of cAMP), a barrier thickness of 1 µm, and a cross-sectional area of 0.3 µm2. The flux coefficient between the microdomain and the cytosol (J12 = 8.0 x 10-16 liters/s) can be better understood by examining the following cases. If the cross-sectional area is the entire surface area of the cell (890 µm2), and the barrier thickness is 1 µm, then J12 can be explained by an effective diffusion coefficient of 9.1 x 10-13 cm2/s. If, instead, the diffusion coefficient is 3 x 10-6 cm2/s and the barrier thickness is 1 µm, then J12 can be explained by a cross-sectional area of 2.7 x 10-3 µm2. It is likely that the barrier consists of both a reduced diffusion coefficient and a cross-sectional area smaller than the entire surface area of the cell. For example, if each one is reduced by the same factor (576), then J12 can be explained by an effective diffusion coefficient of 5.2 x 10-9 cm2/s and a cross-sectional area of 1.5 µm2. A challenge for the future is to determine the size of the microdomains and the exact nature of the permeability barriers.
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DISCUSSION |
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The use of oCNG channels as cAMP sensors has allowed the first real-time, localized measurement of cAMP concentration in single cells. By comparing local and global measures of cAMP accumulation, we conclude that the diffusion of cAMP from the subcellular compartments where it is produced is hindered. This conclusion is reinforced by the observation that forskolin-induced cAMP production can be detected in the rapidly dialyzed, whole-cell configuration. These results are easily described by a three-compartment model (microdomain, cytosol, whole-cell pipette) in which the transfer rates between compartments were determined in independent experiments.
The Advantages of oCNG Channels as cAMP Sensors
We have demonstrated here that oCNG channels give a higher resolution view of cAMP signaling than other available methods. In this paper, we have taken advantage of several of the unique features of oCNG channels: membrane localization, accurate calibration, dynamic range, and lack of desensitization. Moreover, we found that these channels colocalize with AC in discrete regions of the surface membrane, which makes them ideal for studying cAMP signals near their point of generation. The channels also have rapid gating kinetics and, although we have not yet taken advantage of it, this feature will be very useful in studying the kinetics of AC regulation and the dynamics of cAMP signals.
Another way to use oCNG channels as cAMP sensors is to measure Ca2+ influx with optical methods. This would allow the measurement of localized cAMP signals in cells or cellular regions where electrical recording is difficult. For example, localized second messenger signaling has been identified in dendritic spines (
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Three-Dimensional Signaling Domains for cAMP
Previous studies have shown that elements of G protein signaling complexes localize within distinct subdomains of the surface membrane (e.g., caveolae;
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The domains of restricted cAMP diffusion may explain our previous results, which demonstrated that local Ca2+ rises regulate AC activity, whereas global Ca2+ rises have little influence on AC activity (
Physiological Implications of Diffusionally Restricted Microdomains
The hindered diffusion of both Ca2+ and cAMP from these microdomains may play important roles in the regulation of cAMP signaling. Such regions may speed localized cAMP signaling through PKA. A-kinase anchoring proteins (AKAPs) target PKA to specific subcellular regions, and thus facilitate PKA modulation of particular proteins (
These microdomains may also allow for local and precise feedback control of AC. CNG channels could play a role here. Our findings that they functionally colocalize with Ca2+-stimulable AC (type 8 in HEK-AC8 cells) and Ca2+-inhibitable AC (type 6 in C6-2B cells) suggest that Ca2+ influx through these channels provides rapid stimulatory or inhibitory feedback to AC. This may be an important function of CNG channels in nonsensory cells. In this vein, the low apparent cAMP affinities of CNG channels might be explained if they reside primarily in microdomains, where high concentrations can build up. In addition to Ca2+ influx through CNG channels, PKA stimulation of Ca2+ channel activity could also provide feedback regulation of AC. Local Ca2+ feedback regulation of AC could initiate dynamic fluctuations in cAMP (
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Footnotes |
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Portions of this work were previously published in abstract form (Rich, T.C., K.A. Fagan, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 1999. J. Gen. Physiol. 114:16a; Rich, T.C., K.A. Fagan, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. Biophys. J. 78:391A).
1 Abbreviations used in this paper: AC, adenylyl cyclase; AKAP, A-kinase anchoring protein; HEK, human embryonic kidney; NPE-cAMP, 1-(2-nitrophenyl)ethyl-cAMP; oCNG channel, olfactory cyclic nucleotide-gated channel; PDE, phosphodiesterase.
2 The forskolin concentrations used in this study were similar to those used in other studies (1050 µM;
3 These results also serve another important purpose, in that they verify the calibration of the sensor in the whole-cell environment. The time courses of the wash-in of 40 µM and 1 mM cAMP are consistent with a K1/2 value for the channels of 40 µM, and inconsistent with K1/2 values 20 µM. The response to 1 mM cAMP was much faster because high concentrations reached the channel well before the pipette solution equilibrated with the microdomain. If the K1/2 were much lower than 40 µM, simulations show that the wash-in of 1 mM cAMP would be considerably faster than 38 s. The flash photolysis experiment in Fig 1 A is also consistent with K1/2 being > 20 µM. The response to a flash was much larger than the response to 20 µM cAMP (added to the patch pipette). These experiments reinforce the conclusion that high local concentrations of cAMP were generated in response to forskolin stimulation.
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Acknowledgements |
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We thank Dr. R.R. Reed for providing the cDNA encoding the olfactory CNG channel, Dr. J. Cali for providing the HEK-AC8 cell line, and Drs. R.L. Brown, K. Svoboda, and A. Zweifach for helpful comments on the manuscript.
This work was supported by National Institute of Health grants GM32438, NS28389, HL58344, and DC00385.
Submitted: 24 March 2000
Revised: 19 May 2000
Accepted: 1 June 2000
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References |
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Adams, S.R., Harootunian, A.T., Buechler, Y.J., Taylor, S.S., Tsien, R.Y. 1991. Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349:694-697[Medline].
Allen, M.L., Koh, D.S., Tempel, B.L. 1998. Cyclic AMP regulates potassium channel expression in C6 glioma by destabilizing Kv1.1 mRNA. Proc. Natl. Acad. Sci. USA 95:7693-7698
Barber, R., Butcher, R.W. 1980. The quantitative relationship between intracellular concentration and egress of cyclic AMP from cultured cells. Mol. Pharmacol. 19:38-43[Abstract].
Barovsky, K., Pedone, C., Brooker, G. 1983. Forskolin-stimulated cyclic AMP accumulation mediates protein synthesis-dependent refractoriness in C6-2B rat glioma cells. J. Cyclic Nucleotide Res. 9:181-189.
Baylor, D.A., Lamb, T.D., Yau, K.W. 1979. Responses of retinal rods to single photons. J. Physiol. 288:613-634[Abstract].
Beavo, J.A. 1995. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol. Rev. 75:725-748
Bhalla, U.S., Iyengar, R. 1999. Emergent properties of networks of biological pathways. Science 283:381-387
Boyajian, C.L., Garritsen, A., Cooper, D.M.F. 1991. Bradykinin stimulates Ca2+ mobilization in NCB-20 cells leading to direct inhibition of adenylyl cyclase. J. Biol. Chem. 266:4995-5003
Cameron, D.A., Pugh, E.N. 1990. The magnitude, time course and spatial distribution of current induced in salamander rods by cyclic guanine nucleotides. J. Physiol. 430:419-439[Abstract].
Carslaw, H.S., Jaeger, J.C. 1959. Conduction of Heat in Solids. Oxford, UK, Clarendon Press, pp. 510.
Cassano, S., Di Lieto, A., Cerillo, R., Avvedimento, E.V. 1999. Membrane-bound cAMP-dependent protein kinase controls cAMP-induced differentiation in PC12 cells. J. Biol. Chem. 274:32574-32579
Chen, C.H., Nakamura, T., Koutalos, Y. 1999. Cyclic AMP diffusion coefficient in frog olfactory cilia. Biophys. J. 76:2861-2867
Cooper, D.M.F., Karpen, J.W., Fagan, K.A., Mons, N.E. 1998. Ca2+-sensitive adenylyl cyclases. Adv. Second Messenger Phosphoprotein Res. 32:23-51[Medline].
Cooper, D.M.F., Mons, N., Karpen, J.W. 1995. Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature 374:421-424[Medline].
Crank, J. 1975. The Mathematics of Diffusion. Oxford, UK, Clarendon Press, pp. 414.
DeBernardi, M.A., Brooker, G. 1996. Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging. Proc. Natl. Acad. Sci. USA 93:4577-4582
DeBernardi, M.A., Brooker, G. 1998. Simultaneous fluorescence ratio imaging of cyclic AMP and calcium kinetics in single living cells. Adv. Second Messenger Phosphoprotein Res 32:195-213[Medline].
Debernardi, M.A., Munshi, R., Yoshimura, M., Cooper, D.M.F., Brooker, G. 1993. Predominant expression of type-VI adenylate cyclase in C6-2B rat glioma cells may account for inhibition of cyclic AMP accumulation by calcium. Biochem. J. 293:325-328[Medline].
Dessauer, C.W., Gilman, A.G. 1996. Purification and characterization of a soluble form of mammalian adenylyl cyclase. J. Biol. Chem. 271:16967-16974
Dessauer, C.W., Gilman, A.G. 1997. The catalytic mechanism of mammalian adenylyl cyclase equilibrium binding and kinetic analysis of P-site inhibition. J. Biol. Chem. 272:27787-27795
Detlev, S., Restrepo, D. 1998. Transduction mechanisms in vertebrate olfactory receptor cells. Physiol. Rev. 78:429-466
Dhallan, R.S., Yau, K.W., Schrader, K.A., Reed, R.R. 1990. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature 347:184-187[Medline].
Fagan, K.A., Mons, N., Cooper, D.M.F. 1998. Dependence of the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells on capacitative Ca2+ entry. J. Biol. Chem. 273:9297-9305
Fagan, K.A., Rich, T.C., Tolman, S., Schaack, J., Karpen, J.W., Cooper, D.M.F. 1999. Adenovirus-mediated expression of an olfactory cyclic nucleotidegated channel regulates the endogenous Ca2+-inhibitable adenylyl cyclase in C6-2B glioma cells. J. Biol. Chem. 274:12445-12453
Fick, A. 1855. Ueber diffusion. Ann. Phys. Chem. 94:59-86.
Finch, E.A., Augustine, G.J. 1998. Local calcium signalling by inositol-1,4,5-trisphosphate in Purkinje cell dendrites. Nature 396:753-756[Medline].
Finn, J.T., Grunwald, M.E., Yau, K.W. 1996. Cyclic nucleotidegated ion channels: an extended family with diverse functions. Annu. Rev. Physiol. 58:395-426[Medline].
Frace, A.M., Mery, P.-F., Fischmeister, R., Hartzell, H.C. 1993. Rate-limiting steps in ß-adrenergic stimulation of cardiac calcium current. J. Gen. Physiol. 101:337-353[Abstract].
Frings, S., Seifert, R., Godde, M., Kaupp, U.B. 1995. Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotidegated channels. Neuron 15:169-179[Medline].
Gold, G.H. 1999. Controversial issues in vertebrate olfactory transduction. Annu. Rev. Physiol. 61:857-871[Medline].
Gray, P.C., Scott, J.D., Catterall, W.A. 1998. Regulation of ion channels by cAMP-dependent protein kinase and A-kinase anchoring proteins. Curr. Opin. Neurobiol. 8:330-334[Medline].
Gray-Keller, M., Denk, W., Shraiman, B., Detwiler, P.B. 1999. Longitudinal spread of second messenger signals in isolated rod outer segments of lizards. J. Physiol. 513:679-692.
Hagen, V., Dzeja, C., Frings, S., Bendig, J., Krause, E., Kaupp, U.B. 1996. Caged compounds of hydrolysis-resistant analogues of cAMP and cGMP: synthesis and application to cyclic nucleotidegated channels. Biochemistry 35:7762-7771[Medline].
Harootunian, A.T., Adams, S.R., Wen, W., Meinkoth, J.L., Taylor, S.S., Tsien, R.Y. 1993. Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion. Mol. Biol. Cell 4:993-1002[Abstract].
Hille, B. 1992. Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates, Inc, pp. 607.
Horn, R., Marty, A. 1988. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92:145-159[Abstract].
Huang, C., Hepler, J.R., Chen, L.T., Gilman, A.G., Anderson, R.G.W., Mumby, S.M. 1997. Organization of G proteins and adenylyl cyclase at the plasma membrane. Mol. Biol. Cell 8:2365-2378
Jurevicius, J., Fischmeister, R. 1996. cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by ß-adrenergic agonists. Proc. Natl. Acad. Sci. USA 93:295-299
Karpen, J.W., Zimmerman, A.L., Stryer, L., Baylor, D.A. 1988. Gating kinetics of the cyclic-GMPactivated channel of retinal rods: flash photolysis and voltage-jump studies. Proc. Natl. Acad. Sci. USA 85:1287-1291[Abstract].
Koutalos, Y., Brown, R.L., Karpen, J.W., Yau, K.W. 1995a. Diffusion coefficient of the cyclic GMP analog 8-(fluoresceinyl)thioguanosine 3',5' cyclic monophosphate in the salamander rod outer segment. Biophys. J. 69:2163-2167[Abstract].
Koutalos, Y., Nakatani, K., Yau, K.-W. 1995b. Characterization of guanylate cyclase activity in single retinal rod outer segments. J. Gen. Physiol. 106:863-890[Abstract].
Koutalos, Y., Nakatani, K., Yau, K.W. 1995c. The cGMP-phosphodiesterase and its contribution to sensitivity regulation in retinal rods. J. Gen. Physiol. 106:891-921[Abstract].
Koutalos, Y., Nakatani, K., Yau, K.W. 1995d. Cyclic GMP diffusion coefficient in rod photoreceptor outer segments. Biophys. J. 68:373-382[Abstract].
Krupinski, J., Coussen, F., Bakalyar, H.A., Tang, W.J., Feinstein, P.G., Orth, K., Slaughter, C., Reed, R.R., Gilman, A.G. 1989. Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244:1558-1564[Medline].
Lagnado, L., Baylor, D.A. 1992. Signal flow in visual transduction. Neuron 8:995-1002[Medline].
Lamb, T.D., McNaughton, P.A., Yau, K.W. 1981. Spatial spread of activation and background desensitization in toad rod outer segments. J. Physiol 319:463-496[Medline].
Levitan, I.B. 1994. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu. Rev. Physiol. 56:193-212[Medline].
Liu, M., Chen, T.Y., Ahamed, B., Li, J., Yau, K.W. 1994. Calcium-calmodulin modulation of the olfactory cyclic nucleotidegated cation channel. Science 266:1348-1354[Medline].
Lowe, G., Gold, G.H. 1993. Contribution of ciliary cyclic nucleotidegated conductance to olfactory transduction in the salamander. J. Physiol. 462:175-196[Abstract].
Ma, H.T., Patterson, R.L., van Rossum, D.B., Birnbaumer, L., Mikoshiba, K., Gill, D.L. 2000. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science 287:1647-1651
Molday, R.S. 1998. Photoreceptor membrane proteins, phototransduction, and retinal degenerative diseases: the Friedenwald lecture. Invest. Opthalmol. Vis. Sci. 39:2493-2513.
Mons, N., Harry, A., Dubourg, P., Premont, R.T., Iyengar, R., Cooper, D.M.F. 1995. Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses. Proc. Natl. Acad. Sci. USA 92:8473-8477[Abstract].
Montminy, M. 1997. Transcriptional regulation by cyclic AMP. Annu. Rev. Biochem. 66:807-822[Medline].
Nakatani, K., Yau, K.-W. 1988. Guanosine 3',5'-cyclic monophosphateactivated conductance studied in a truncated rod outer segment of the toad. J. Physiol. 395:731-753[Abstract].
Naraghi, M., Neher, E. 1997. Linearized buffered Ca2+ diffusion in microdomains and its implications for calculation of [Ca2+] at the mouth of a calcium channel. Biophys. J. 17:6961-6973.
Okamoto, T., Schlegel, A., Scherer, P.E., Lisanti, M.P. 1998. Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J. Biol. Chem. 273:5419-5422
Olson, A., Pugh, E.N. 1993. Diffusion coefficient of cyclic GMP in salamander rod outer segments estimated with two fluorescent probes. Biophys. J. 65:1335-1352[Abstract].
Patterson, R.L., van Rossum, D.B., Gill, D.L. 1999. Store-operated Ca2+ entry: evidence for a secretion-like coupling model. Cell 98:487-499[Medline].
Polans, A., Baehr, W., Palczewski, K. 1996. Turned on by Ca2+! The physiology and pathology of Ca2+-binding proteins in the retina. Trends Neurosci. 19:547-554[Medline].
Pugh, E.N., Duda, T., Sitaramayya, A., Sharma, R.K. 1997. Photoreceptor guanylate cyclases: a review. Biosci. Rep. 17:429-473[Medline].
Pugh, E.N., Lamb, T.D. 1993. Amplification and kinetics of the activation steps in phototransduction. Biochim. Biophys. Acta. 1141:111-149[Medline].
Purves, R.D. 1977. The time course of cellular responses to iontophoretically applied drugs. J. Theor. Biol. 65:327-344[Medline].
Pusch, M., Neher, E. 1988. Rates of diffusional exchange between small cells and a measuring patch pipette. Pflügers Arch. 411:204-211.
Smit, M.J., Iyengar, R. 1998. Mammalian adenylyl cyclases. Adv. Second Messenger Phosphoprotein Res 32:1-21[Medline].
Stryer, L. 1991. Visual excitation and recovery. J. Biol. Chem. 266:10711-10714
Sudlow, L.C., Gillette, R. 1997. Cyclic AMP levels, adenylyl cyclase activity, and their stimulation by serotonin quantified in intact neurons. J. Gen. Physiol. 110:243-255
Takechi, H., Eilers, J., Konnerth, A. 1998. A new class of synaptic response involving calcium release in dendritic spines. Nature 396:757-760[Medline].
Tian, D., Huang, D., Brown, R.C., Jungmann, R.A. 1998a. Protein kinase A stimulates binding of multiple proteins to a U-rich domain in the 3'-untranslated region of lactate dehydrogenase A mRNA that is required for the regulation of mRNA stability. J. Biol. Chem. 273:28454-28460
Tian, D., Huang, D., Short, S., Short, M.L., Jungmann, R.A. 1998b. Protein kinase A-regulated instability site in the 3'-untranslated region of lactate dehydrogenase-A subunit mRNA. J. Biol. Chem. 273:24861-24866
Trautwein, W., Hescheler, J. 1990. Regulation of the L-type calcium current by phosphorylation and G proteins. Annu. Rev. Physiol. 52:257-274[Medline].
Trivedi, B., Kramer, R.H. 1998. Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists. Neuron 21:895-906[Medline].
Tsien, R.W. 1983. Calcium channels in excitable cell membranes. Annu. Rev. Physiol. 45:341-358[Medline].
Wootton, J.F., Trentham, D.R. 1989. "Caged" compounds to probe the dynamics of cellular processes: synthesis and properties of some novel photosensitive P-2-nitrobenzyl esters of nucleotides. NATO ASI Ser. Ser. C Math. Phys. Sci. 272:277-296.
Yarfitz, S., Hurley, J.B. 1994. Transduction mechanisms of vertebrate and invertebrate photoreceptors. J. Biol. Chem. 269:14329-14332
Yau, K.W. 1994. Phototransduction mechanism in retinal rods and cones: the Friedenwald lecture. Invest. Ophthalmol. Vis. Sci. 35:9-32[Medline].
Zaccolo, M., De Giorgi, F., Cho, C.Y., Feng, L., Knapp, T., Negulescu, P.A., Taylor, S.S., Tsien, R.Y., Pozzan, T. 2000. A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell Biol. 2:25-29[Medline].
Zhou, Z., Neher, E. 1993. Mobile and immobile calcium buffers in bovine adrenal chromaffin cells. J. Physiol. 469:245-273[Abstract].
Zweifach, A., Lewis, R.S. 1993. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc. Natl. Acad. Sci. USA 90:6295-6299[Abstract].