Correspondence to: Anita L. Zimmerman, Department of Molecular Pharmacology, Physiology and Biotechnology, Box G-B329, Brown University, Providence, RI 02912. Fax:(401) 863-1222 E-mail:anita_zimmerman{at}brown.edu.
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Abstract |
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Cyclic nucleotidegated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric ( subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (Po), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the Po but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayerchannel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.
Key Words: rod, olfactory receptor, channel modulation, lipid bilayer, tetracaine
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INTRODUCTION |
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Cyclic nucleotidegated (CNG)1 ion channels play a vital role in visual transduction (for review see
In rod cells, light activates an enzyme cascade that decreases the level of cytosolic cGMP, resulting in the closure of CNG channels that are normally open in the dark (for review see
We previously reported that DAG modulation of the native CNG channel from frog rod outer segments is independent of phosphorylation ( and ß subunits) expressed in Xenopus oocytes has shown that long-chain DAG is stimulatory, whereas the cellular precursor to DAG, PIP2, is inhibitory. However, PIP2 inhibition is not as strong when only
subunits are expressed (
To elucidate the mechanism of DAG inhibition of CNG channels, we explored the effect of a short-chain DAG analogue on cloned rod and olfactory channels expressed in Xenopus oocytes. Rod channels exhibited greater inhibition than olfactory channels at saturating cGMP concentrations. However, DAG inhibition was much more efficient at low open probabilities for both channel types, suggesting that DAG stabilizes the closed states of the channel. In addition, the Hill coefficients from DAG doseresponse curves suggested that multiple DAG molecules participate in the inhibition of a channel.
Because these two CNG channels showed differences in their inhibition by DAG, we also examined the DAG modulation of a series of chimeras of the rod and olfactory channels (1 and shows obvious voltage dependence (
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MATERIALS AND METHODS |
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Expression of Channels in Xenopus Oocytes
The plasmids containing the subunits of bovine rod (CNG1), rat olfactory (CNG2), and chimeric cDNA were provided by William N. Zagotta (University of Washington, Seattle, WA). See
subunit clone originated from the laboratories of R.R. Reed (Johns Hopkins University, Baltimore, MD) and R.S. Molday (University of British Columbia, Vancouver, British Columbia, Canada). Construction of the chimeras was previously described in
Sections of ovaries containing oocytes were surgically removed from anesthetized Xenopus laevis frogs, and individual oocytes were isolated from clumps of tissue by treatment with 1 mg/ml collagenase type 1A (Sigma-Aldrich) in a buffer containing low calcium (82.5 mM NaCl, 2.5 mM KCl, 5 mM HEPES, and 1 mM MgCl2, pH 7.6). The cRNA was injected into Xenopus oocytes using a Drummond Nanoject oocyte injector. The injected oocytes were initially incubated at 15°C overnight, and then at 18°C for 110 d before testing for expression via the patch-clamp technique. For single channel studies, the oocytes were stored at 4°C once a desirable level of expression was reached. The saline solution for maintaining oocytes contained the following: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, 1 mM MgCl2, 2.5 mM pyruvic acid, 100 U/ml penicillin, and 100 µg/ml streptomycin, pH 7.6. The vitelline membrane was removed by treatment with a hypertonic solution (200 mM N-methyl-D-glucamine, 2 mM KCl, 10 mM EGTA, 10 mM HEPES, and 1 mM MgCl2, pH 7.4) and mechanical dissection to expose the plasma membrane for patch-clamp recording.
Application of Electrophysiological Solutions
The oocyte chamber for patch-clamp experiments was a plastic petri dish, and water-soluble solutions were applied using a 36-solution patch perfusion system, the RSC-100 rapid solution changer (Molecular Kinetics). Solutions consisted of the following: 130 mM NaCl, 0.2 mM EDTA, and 2 mM HEPES buffer, pH 7.2, with varying concentrations of cGMP (Sigma-Aldrich) and cAMP (Calbiochem-Novabiochem">Calbiochem-Novabiochem). When necessary, 500 µM niflumic acid (Sigma-Aldrich) was added to the extracellular solution to block Ca2+-activated Cl- channels, which are endogenous to the oocyte membranes. The DAG analogue, 1,2-dioctanoyl-sn-glycerol (1,2-DiC8; Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) at 0.520-mM stock concentrations and stored at -20°C. Because of the lipophilicity of the analogues, the DAG solutions were not applied to the patch through Teflon tubing because we found that this resulted in a decrease in the response, as though the DAG had adhered to the tubing, effectively reducing the concentration of the DAG before its exposure to the patch. Instead, the DAG analogue was applied to the intracellular surfaces of patches by direct addition to the bathing solution in the petri dish, where it was carefully and thoroughly mixed using a transfer pipet. The petri dish and agar bridge were replaced after each experiment with DAG.
Electrophysiological Recordings and Analysis
Data were collected from inside-out patches in the steady state after any spontaneous changes in cGMP affinity, which usually resulted in a left shift of the cGMP doseresponse curve. These changes have been attributed to dephosphorylation by endogenous patch-affiliated phosphatases (10 to 40 min, and the magnitude and time course of the effect varied with the channel type.
For DAG doseresponse curves, the bath contained a saturating concentration of cGMP (2 mM for rod and chimeric channels and 100 µM for olfactory), and DAG was added in increments resulting in changes in bath concentration ranging from 0.05 to 48 µM. However, the DMSO concentration remained below 1.2%, which had no effect on the channels or the leak current. The current was monitored for several minutes after each addition of DAG to insure that the effect of the DAG had stabilized; stabilization was usually complete within <1 or 2 min. This time may have been required for DAG to insert into the membrane, and the reversal of the DAG effect was also slow as reported previously (
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For experiments with the native channel, rod cells were obtained from Rana Pipiens according to an established method (. All recordings were obtained at room temperature (2025°C). Recordings of the current were made as the voltage was changed from -100 to +100 mV in steps of 20 or 50 mV, from a holding potential of 0 mV. The leak currents obtained in the absence of cGMP were subtracted from each record. For single-channel studies, three to five continuous 1-s recordings were made at +80 mV.
Sampling (digitization) of currents was accomplished using an Axopatch 1B, 1C, or 200 patch-clamp amplifier (Axon Instruments) with analogue-to-digital converters, which sent output to a Macintosh Quadra computer running Pulse software (Instrutech). Before digitization, the data were low passfiltered using a selectable 8-pole Bessel filter (Frequency Devices). The filter cutoff frequency (-3 dB point) was 2 kHz for multichannel patches and 5 kHz for single-channel patches. Sampling rates were set at five times the filter cutoff frequency to prevent aliasing. Data analysis was performed with the graphical analysis software IgorPro (WaveMetrics).
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RESULTS |
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DAG Inhibition of Rod Channels Is Greater than that of Olfactory Channels
Fig 1 demonstrates that DAG has a greater inhibitory effect on rod channels than on olfactory channels and that both channel types are more sensitive to DAG at low concentrations of agonist. Electrophysiological recordings were obtained from inside-out excised patches from Xenopus oocytes expressing homomultimers of bovine rod or rat olfactory CNG ion channels. Fig 1 shows the current traces recorded for each wild-type channel at low and high cGMP concentrations in both the presence and absence of 0.5 µM DAG. At saturating cGMP concentrations, this low dose of DAG had a small effect on the olfactory channel, but decreased the current of the rod channel by
42%. At lower cGMP concentrations (near the K1/2 for each channel), the same amount of DAG reduced the current for both rod and olfactory channels to a greater extent. This suppression was found to be reversible (data not shown), which is consistent with the reversible suppression observed for the native rod channels (
Fig 2 illustrates a more quantitative comparison of the response of the two channels to DAG. Doseresponse curves for activation of each channel by cGMP demonstrate a large shift to the right in the presence of 0.5 µM DAG. For the rod channel, the K1/2 increased by a factor of 3.2, from 24 to 76 µM cGMP, and for the olfactory channel, the K1/2 increased by a factor of 2.2, from 1.8 to 4.0 µM cGMP. For the rod channel, there is also a significant (29%) reduction in the maximal current with DAG, whereas this reduction is much smaller (3%) for the olfactory channel. The Hill coefficients remained essentially unchanged for both channels.
Previous results showed that application of excess cyclic nucleotide did not reverse the inhibition of the native rod channel by DAG, suggesting that the lower apparent affinity does not result from competitive inhibition of agonist binding (
The doseresponse curves for DAG at saturating [cGMP] are dramatically different for the rod and olfactory channels. Fig 3 indicates that rod cGMP-activated currents are completely suppressed by 3 µM DAG at saturating (2 mM) cGMP, but the olfactory cGMP-activated currents show only partial suppression at saturating (100 µM) cGMP, even with DAG concentrations as high as 12 µM. In fact, the fractional suppression of the olfactory channel remained at this low level even when we raised the DAG concentration to 48 µM (data not shown). For the rod channel, the IC50 for inhibition was 0.83 µM DAG, and the Hill coefficient was 1.7. However, for the olfactory channel, the concentration required to obtain half maximal inhibition was 3.24 µM DAG, with a Hill coefficient of 1.4. Thus, there is approximately a fourfold difference between the DAG IC50 obtained for the rod channel and that obtained for the olfactory channel at saturating [cGMP].
Fig 3 also demonstrates the higher sensitivity of both channels to DAG at lower concentrations of cGMP. The DAG doseresponse curve from a representative patch at low [cGMP] is shown for each channel. In comparison to the corresponding DAG doseresponse curve at high [cGMP], the curve at low [cGMP] is shifted to the left for both channels, and, interestingly, for the olfactory channel, the inhibition reaches 100%. The I/Imax for both channels was 0.40 before the addition of DAG; the IC50 values for inhibition were 0.41 and 0.47 µM, and the Hill coefficients were 1.3 and 1.4 for the rod and olfactory channels, respectively. Furthermore, for the rod channel, DAG inhibition at saturating [cAMP] was comparable to that at a low cGMP concentration that produced a similar fractional activation (data not shown). This finding suggests that DAG preferentially affects closed channels, regardless of ligand occupancy.
For the native rod channel, the DAG IC50 was previously reported to be 22 µM, which is quite different from the value reported here for the cloned rod homomultimer. Although preliminary studies with the cloned channel resulted in development of a new method of administering DAG to the patch without the use of Teflon (see MATERIALS AND METHODS), we could not ignore the possibility that the cloned channel might be more sensitive to the DAG analogue than is the native channel. Thus, we reexamined the native channel using our new DAG application technique. Our data confirmed that the response of the native channel to the DAG analogue is similar to that for the cloned channel, as shown by the triangles in Fig 3. This indicates that the values from the previous study are in error, most likely because of the adherence of DAG to the Teflon delivery tubes, effectively reducing the concentration of DAG in solution.
Reductions in Maximal Current Are Due to Decreasing Open Probability
Single-channel analysis was performed to determine whether the reduction in the maximal current by DAG was due to a decrease in open probability (Po) or in single-channel conductance. Fig 4 and Fig 5 show single-channel currents and amplitude histograms at +80 mV for rod and olfactory channels in the presence and absence of DAG. The patch in Fig 4 contained two rod channels (see legend for note about sequence); the patch in Fig 5 contained a single wild-type olfactory channel. In the presence of saturating cGMP, both rod and olfactory channels spent most of their time in the open state. With the addition of DAG to the bath, both types of channels spent more time closed. Furthermore, the single-channel amplitude remained unchanged by the DAG, at 2.1 pA for the rod channel and 3.2 pA for the olfactory channel. Thus, DAG causes a reduction in Po, but does not appear to change the single-channel conductance.
DAG Inhibition Is Independent of Voltage
The addition of DAG at saturating cGMP alters the steady state currentvoltage relations of both channels in a manner that reflects the lower Po of each channel in the presence of DAG, suggesting that DAG does not impart its own voltage dependence but merely conveys the gating behavior that is apparent when fewer channels are open. In Fig 6, the currentvoltage relations for each channel are shown under three different experimental conditions: at saturating cGMP, both in the presence and absence of 2 µM DAG, as well as at a low cGMP concentration. For the rod channel, the addition of DAG at saturating cGMP creates a currentvoltage relation similar to that observed when the channel responds to 10 µM cGMP, which is below the K1/2 value for this patch. These relations show the characteristic increase in rectification seen for the native rod channel at low cGMP concentrations (
Structural Localization of Channel Region(s) Influencing Differential DAG Inhibition of Rod and Olfactory Channels
We used chimeras of rod and olfactory channels in an attempt to identify channel regions responsible for the differential inhibition of these two channels by DAG. For each chimera, we measured the K1/2 for activation by cGMP, the maximal percent inhibition by DAG at saturating cGMP and the DAG IC50 for this level of inhibition. Data were averaged from three to six patches for each chimera. Fig 7 shows DAG doseresponse curves measured at saturating cGMP for three representative chimeras, CHM3, CHM11, and CHM13 (see Fig 8 for structures). Interestingly, although CHM3 contains the amino and carboxyl termini of the rod channel, its response to DAG is more like that of the olfactory channel; it is only partially inhibited by DAG and its IC50 (3.30 µM) is closer to that of the olfactory channel than to that of the rod channel. These data provided the first hint that the termini are not as important as the transmembrane segments and loops in determining the differential effects of DAG on rod and olfactory channels. Additional support for this notion comes from experiments with a mutant of the rod channel, H468Q. This mutant channel has dramatically increased cAMP efficacy (with saturating cAMP producing 40% of the maximal cGMP-activated current, as compared with only one to a few percent for wild-type rod channels). However, its DAG IC50 (0.33 µM, mean from 11 patches; data not shown) is not very different from that of the wild-type rod channel, and it is fully inhibited by DAG at saturating cGMP. Thus, functionally significant mutations in the carboxyl terminus have rather insignificant effects on inhibition by DAG.
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Fig 8 shows the values of cGMP K1/2 and of DAG IC50 at saturating cGMP for each chimera and the two wild-type channels. The results are displayed, along with the chimera structures, in the order of increasing cGMP K1/2, which reflects the relative ease of opening of each chimera. Similar to the data for tetracaine inhibition observed by
The corresponding maximum inhibition by DAG at saturating cGMP for each chimera and the wild-type channels is shown in Fig 9, presented in the order of increasing maximum inhibition. The chimeras that reveal only partial inhibition tend to contain more olfactory-like sequences in the core regions of the channel, from S2 through S6 including the interconnecting loops of the transmembrane segments. In general, differences in the amino and carboxyl termini do not explain the differential DAG inhibition of the rod and olfactory channels; however, it appears that the presence of both amino and carboxyl termini from the rod in CHM3 may influence this chimera's behavior, increasing the maximum inhibition toward that of the rod channel.
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DISCUSSION |
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Although extensive research has been performed on CNG channels, the molecular mechanism of gating is not fully understood. By exploring the molecular mechanism of DAG inhibition of these channels, we may further understand the complex gating of these ion channels and, in particular, the differences between the properties of the rod and olfactory channels. In this study, we have compared DAG inhibition of the bovine rod and rat olfactory homomultimeric channels expressed in Xenopus oocytes. For both channels, inhibition by DAG is greater at low than at high channel Po, indicating that DAG stabilizes the closed states of the channels (Fig 1 and Fig 3). This finding is consistent with that previously reported for DAG inhibition of the native CNG channel in amphibian rod cells (
We find that rod CNG channels are much more strongly inhibited by DAG than are olfactory channels. This is especially apparent at saturating cGMP concentrations, where the rod cGMP-activated currents are completely abolished by DAG, but the olfactory currents are only partially suppressed even at high concentrations of DAG (Fig 3). This finding suggests that the olfactory channel, whose opening is more energetically favored (
Our previous work with the native rod CNG channel suggested that DAG may be a closed state inhibitor (
To explore further the behavior of the channels in response to DAG, we also examined patches containing only one or two channels. Single-channel analysis revealed a DAG-induced decrease in Po without a measurable change in single-channel conductance (Fig 4 and Fig 5). Hence, the reduction in the maximum current at saturating cGMP most likely was not due to the incomplete block of the pore by DAG, resulting in channel openings of lower current amplitude. The Hill coefficients from the DAG doseresponse curves suggest that if DAG were to act by blocking the pore, it must do so with more than 1 molecule per channel. It appears that at least two DAG molecules are necessary for efficient inhibition of each channel (Fig 3), raising the question of whether one DAG molecule interacts with each of the four channel subunits.
Since cloned rod and olfactory channels are differentially inhibited by DAG, we used a series of chimeras of these two channels in an attempt to attribute these differences in inhibition to specific regions of the channels. Chimeras have offered a valuable approach to understanding the differences in rod and olfactory channel behavior with respect to apparent agonist affinity, efficacy, and tetracaine inhibition (
Since PKC activation is not involved, DAG may have a direct inhibitory interaction with the CNG channels themselves or act on an unknown channel modulator that does not require the addition of nucleoside triphosphates. A previously defined DAG binding motif in protein kinase C and RasGRP contains repeats of histidine and cysteine residues (
Our chimera study suggests that the transmembrane segments and attached loops may be important in determining the differential effects of DAG on rod and olfactory channels. Given the hydrophobic nature of DAG, the transmembrane segments are a likely target for this inhibitor. Furthermore, the spatial arrangement of the transmembrane segments may be critical for proper channel function. In other channels, rotation or twisting of these helical segments has been proposed for the allosteric transition from the closed to open state (
In Fig 10, we present two working models that represent possible mechanisms of DAG inhibition of olfactory and rod channels. Both models depict the two channels in either open or closed configurations (with and without DAG) at saturating agonist concentrations. The horizontal transitions in each model represent allosteric state changes, whereas vertical transitions represent binding or removal of multiple molecules of DAG. The top part of each diagram portrays the preference of both channel types for the open state in the absence of DAG, and the tendency for rod channels to be closed more of the time than are olfactory channels. For the rod channel, DAG strongly stabilizes the closed state, even under saturating agonist conditions. However, for the olfactory channel, the transition to the open state is still highly favored, even in the presence of DAG. This representation is based on the data showing that even high concentrations of DAG can only partially inhibit the olfactory channel at saturating [cGMP] (Fig 3).
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In the first model (Fig 10 A), DAG is shown bound to a putative crevice on the intracellular side within the hydrophobic core of the channel. Alternatively, DAG may directly bind the channel at the hydrophobic interface between the transmembrane segments and the bilayer. An equally plausible mechanism (Fig 10 B) for the action of DAG is that it inserts into the lipid bilayer, perturbing the coupling between the hydrophobic core of the bilayer and the hydrophobic membrane-spanning regions of the channel. This kind of alteration has been shown to modulate other channels (
The cartoon in Fig 10 B shows one possible scenario by which DAG may alter bilayerchannel interactions. Here, the closed states are drawn distinctly shorter than the open states to suggest a smaller hydrophobic exterior surface of the protein. The surrounding bilayer is drawn with more curvature in the presence of DAG, to suggest a thinner, better matched, hydrophobic core at the interface between the bilayer and the closed channel. Thus, the addition of DAG is predicted to change the energetics of the channel conformational changes, thereby altering the open and closed probabilities. Of course, DAG may regulate the channels by some combination of the mechanisms depicted in Fig 10 (A and B). The actual physical changes that occur in channel gating remain to be determined, as does the detailed mechanism by which DAG alters the conformational equilibrium. However, it would seem that hydrophobic inhibitors such as DAG may prove useful in reporting intramolecular interactions and movements in the hydrophobic core of channels.
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Footnotes |
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1 Abbreviations used in this paper: CNG, cyclic nucleotidegated; DAG, diacylglycerol; Po, open probability.
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Acknowledgements |
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We thank Drs. Sharona Gordon and Gary Yellen for helpful discussions, and Dr. William N. Zagotta for comments on an earlier version of the manuscript. We thank Dr. Maria Ruiz for the mouse olfactory clone and Dr. William N. Zagotta for the bovine rod and rat olfactory clones and the series of chimeric constructs. We are also grateful to Roberto Neisa and Melody Chang for technical assistance, and Joe Zimmerman for software development.
This work was supported by grants from the American Heart Association of Rhode Island (No. 97-07721S) and the National Institutes of Health (No. EY07774).
Submitted: 10 May 2000
Revised: 22 September 2000
Accepted: 13 October 2000
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