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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We previously found that native cyclic nucleotidegated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755768; in this issue). Here, we report that a point mutation at position 204 in the S2S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.
Key Words: rod, olfactory, heart, diacylglycerol, tetracaine
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INTRODUCTION |
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Cyclic nucleotidegated (CNG)1 cation channels are best characterized in the visual and olfactory systems, but they are also present in tissues as diverse as the heart (
The Ih channel in the sinoatrial node of the heart controls the pacemaking activity of the heart and is regulated by the binding of cAMP (
We had previously shown that DAG reversibly inhibits the native rod channel without a phosphorylation reaction as if by direct interaction with some site on the channel (
Several studies have highlighted key regions that influence channel gating. These include the amino and carboxyl termini, which have been proposed to interact during gating, as well as the C-linker, which is located between the last transmembrane segment and the cyclic nucleotide binding domain and may function as a sensor of the binding event that triggers the allosteric opening transition of the channel (
While exploring the mechanism of inhibition by the local anesthetic, tetracaine,
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MATERIALS AND METHODS |
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Mutagenesis and Expression of Channels by Xenopus Oocytes
The plasmids containing the subunits of Brod (CNG1) and Rolf (CNG2) cDNA were generous gifts from W.N. Zagotta (University of Washington, Seattle, WA) and the mouse olfactory (Molf G204E) clone was provided by M.L. Ruiz (Entelos, Inc., Menlo Park, CA) (
The primary sequences of the Molf G204E and Rolf clones were aligned for comparison to identify residue differences, and through mutagenesis and functional analysis, we confirmed that the residue at position 204 was responsible for the differential inhibition by DAG reported in this study. Point mutations were incorporated using either the TransformerTM site-directed mutagenesis kit from CLONTECH Laboratories, Inc. or the QuickChangeTM site-directed mutagenesis kit from Stratagene. Both kits allow mutagenesis in our high expression vector without subcloning. The mutagenic primers were designed to encode the desired mutation and its surrounding sequence in which we also incorporated a restriction enzyme site (for initial screening purposes) without altering the primary sequence of the protein. For the conversion of the Molf G204E to wild-type Molf, the restriction enzyme site that had been introduced during cloning (SacI) was removed to convert the glutamate residue at position 204 to glycine, which was reported in the genomic sequence (
Electrophysiological Methods
All experiments were performed on inside-out membrane patches excised from Xenopus oocytes that were expressing homomultimers of one of the wild-type or mutant channel types. The cell chamber in which patches were excised consisted of a petri dish containing 15 ml of solution with or without saturating concentrations of cGMP. The solution compositions and method of application are as described in the companion article (
Patch-clamp electrodes were prepared from borosilicate glass capillaries with openings that ranged from 0.5 to 20 µm in diameter, producing resistances of 0.615 M. Patches were typically excised in low divalent sodium solutions containing saturating concentrations of cGMP (2 mM for Brod channels and 100 µM for Molf and Rolf channels). A low divalent sodium solution without cGMP was applied as a control to measure the resulting leak current which was subtracted from experimental measurements to obtain the cyclic nucleotideactivated current. We monitored the patch for
1040 min, until patch responses to low agonist concentrations stabilized, to ensure that any spontaneous changes in channel behavior, such as those caused by dephosphorylation (
Patch-clamp data acquisition and analysis were as described in the companion article (
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RESULTS |
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Residue at Position 204 Dictates the Response of Olfactory Channels to DAG
Sequence comparison of the Molf and Rolf clones showed differences at 12 positions throughout the protein, and there were an extra five amino acids located at the carboxyl-terminal tail of the Molf clone. Analysis of the Molf channel cDNA sequence reported in the Entrez database (National Center for Biotechnology Information) revealed that 2 of the 12 changes were introduced with restriction enzyme sites during cloning. The first of these two was a methionine changed to a valine at position 2, and the second was a glycine changed to glutamate at position 204. The other 10 differences appear to be true discrepancies between the Molf and Rolf CNG channels. As will be described in more detail later, the original Molf clone (referred to as Molf G204E) was much more sensitive to DAG than was the wild-type Rolf channel. A concurrent study used chimeras of the Brod and Rolf CNG channels to locate the regions of the Brod channel that could convey the higher sensitivity to DAG; the transmembrane segments and their connecting loops were identified as sensitive regions in the Brod channel (
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Site-directed mutagenesis was used to substitute the glutamate residue at this position with the wild-type glycine residue. This construct will be herein referred to as Molf, the true wild-type channel. To eliminate potential effects of the 11 other differences, the glycine at position 204 in the Rolf channel was replaced with a glutamate residue, permitting evaluation of the effect of this isolated point mutation on the DAG sensitivity of the Rolf channel. The requirement of a negative charge in conveying the observed functional changes was tested by substituting another negatively charged residue (aspartate [D]), or a bulky hydrophobic residue (tryptophan [W]). Residue 204 was also replaced by the positively charged residue lysine (K), but we never observed functional expression with this construct. Fig 1 shows the sequence alignment of 11 homologous CNG channels in the region equivalent to residues 195218 of the Rolf channel. Notably, the glycine at position 204 is conserved throughout these sequences, including in the Brod channel, which is only 55% identical to the Rolf channel, and must have other differences that cause its relatively high sensitivity to DAG.
Apparent Agonist Affinities and cAMP Efficacies of Molf G204E and Rolf G204E CNG Channels Are Similar to those of Wild-type Molf and Rolf Channels
Doseresponse curves for activation by cGMP and cAMP in Fig 2 demonstrate relative apparent agonist affinities for Brod, Molf, Rolf, Molf G204E, and Rolf G204E CNG homomultimeric ( only) channels. The cGMP doseresponse curve for Brod channels is shifted to the right with respect to all other channels shown, indicating its lower apparent affinity for cGMP (Fig 2 A). Wild-type and mutant (G204E) rat and mouse olfactory channels respond with similar relative apparent affinities for cGMP and cAMP (Fig 2A and Fig B). Furthermore, saturating concentrations of both cAMP and cGMP, which act as full agonists, elicit maximal responses from all olfactory channels shown. In contrast, the Brod CNG channel responds with a dramatically higher apparent affinity for cGMP than for cAMP, which acts as only a partial agonist of the channel, activating only a fraction (5%) of the current produced by saturating levels of cGMP, even at maximal cAMP concentrations. Therefore, the sequence differences, including the mutation at residue 204, between the Molf G204E and Rolf channels do not significantly affect the apparent agonist affinities or efficacies of the channels. For the Rolf G204D mutant, the responses to cGMP and cAMP are similar to that of Rolf G204E (data not shown).
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Brod, Molf G204E, and Rolf G204E Channels Display Higher Sensitivity to DAG than do the Wild-type Molf and Rolf Channels
Application of DAG to the intracellular surfaces of excised patches suppressed current activated by cGMP in all CNG channels studied in the absence of ATP, which indicates that the inhibition does not depend on phosphorylation by PKC. The extent of inhibition by DAG varied according to the concentration of agonist and the channel type. Fig 3 demonstrates that the addition of DAG produced a greater inhibition of Brod and Molf G204E channels than of Rolf CNG channels. The current traces were recorded for each channel type at saturating cGMP levels in both the presence and absence of DAG. The addition of 1.5 µM DAG had a large effect on both the Brod and Molf G204E channels, decreasing the maximal current by >80% in each case. However, this dose of DAG had a small effect on the Rolf channel, reducing its maximal current by only 14%. As seen with the native rod CNG channel (
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To further investigate the differences observed in Fig 3, we measured DAG doseresponse curves for each channel type at saturating concentrations of cGMP. Fig 4 illustrates that cGMP-activated currents in Brod, Molf G204E, and Rolf G204E channels are fully suppressed by 3 µM DAG, whereas the cGMP-activated currents of the wild-type Molf and Rolf channels are much less sensitive to the inhibitor, displaying only partial inhibition at 12 µM DAG. Thus, the DAG doseresponse relations of Molf G204E and Rolf G204E channels closely resemble those of Brod CNG channels (Fig 4), even though their apparent affinities for cGMP and cAMP, as well as their cAMP efficacies (Fig 2), resemble those of the wild-type olfactory channels (summarized in Table 1). As discussed later, other gating properties (voltage dependence and kinetics) of these mutant channels were more clearly different from those of the wild-type olfactory channels. As expected, the behavior of the Rolf G204D mutant was found to resemble that of Rolf G204E (data not shown).
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The Mutant (G204E) Olfactory Channels Show Voltage-dependent Gating Not Observed with Wild-type Olfactory Channels
Since the Molf and Molf G204E channels had very different responses to DAG, it was surprising that our initial measurements of gating by cyclic nucleotides seemed so similar. On closer inspection, we found that, unlike wild-type olfactory channels, the Molf G204E and Rolf G204E channels exhibited prominent voltage-dependent gating kinetics similar to those previously described for the native rod CNG channel (
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We also looked for evidence of voltage-dependent gating by plotting doseresponse curves for cGMP at both +100 and -100 mV. Results from multiple patches are shown in Fig 6 for Brod, Rolf, and Rolf G204E. To control for patch-to-patch variability in the apparent cGMP affinity, all data for each patch were normalized to the K1/2 at +100 mV for that patch. For Brod and Rolf G204E, the shift in the data at -100 mV relative to +100 mV indicates voltage-dependent gating. In contrast, the doseresponse curves for the Rolf channel did not shift, suggesting that gating of this channel is not voltage-dependent. The ratios of K1/2 at -100 mV to K1/2 at +100 mV are 1.4 and 1.7 for the Brod and Rolf G204E channels, respectively.
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The Rolf G204W Mutant Demonstrates Decreased Apparent Agonist Affinities and cAMP Efficacy as well as Voltage-Dependent Gating and Higher Sensitivity to DAG
The families of currents in Fig 7 A reveal observable gating kinetics for the Rolf G204W mutant channel that resemble those seen with the Brod channel but not with the wild-type Rolf channel (Fig 5). In addition, Fig 7 (B and C, and Table 1) shows that the apparent agonist affinities (K1/2 values for cGMP and cAMP) of Rolf G204W are intermediate between those for the Rolf and Brod channels (Fig 2 and Table 1). Similarly, the cAMP efficacy for Rolf G204W lies between that for the Rolf and Brod channels. The points in Fig 7 B provide cGMP doseresponse data at two different voltages (+100 and -100 mV), illustrating the voltage dependence of gating that was observed for the Brod channel but not for the Rolf channel (Fig 6). Fig 8 A and Table 1 indicate that DAG inhibition of the Rolf G204W mutant is even more like that of the Brod channel than are the agonist affinities and efficacies; in fact, the Rolf G204W channel is actually more sensitive to DAG than is the Brod channel. These data show that the introduction of a bulky hydrophobic residue (tryptophan) at position 204 in the Rolf channel has an even greater impact on channel function than the introduction of negatively charged residues. In Fig 8 B, the currentvoltage relation for the Rolf G204W channel again shows the slight voltage dependence of the mutant that is intrinsic to gating. As observed for the Brod and Rolf channels (Crary et. al, 2000, in this issue), the addition of DAG does not introduce any further voltage dependence than that which is expected at lower open probability (
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Residue 204 Mutants Are Also More Sensitive to Block by Tetracaine
To explore the mechanism by which residue 204 alters DAG sensitivity, we also studied the very different inhibitor, tetracaine. Tetracaine doseresponse curves (data not shown), obtained for some of the mutant channels at saturating cGMP concentrations, indicated that the sensitivity to tetracaine (like that to DAG) is much greater for Molf G204E and Rolf G204D (IC50 = 10.8 µM for both mutants) than for the Rolf channel (IC50 = 139.6 µM). Thus, the response to tetracaine for both mutants was more like that reported for the Brod channel (IC50 = 2.6 µM;
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DISCUSSION |
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We have identified a residue in the S2S3 loop that is highly conserved among the CNG channel family members, and that influences channel gating and inhibition by DAG and tetracaine. Replacement of the glycine at position 204 of the mouse or rat olfactory channel with a negatively charged residue (glutamate or aspartate) created channels with dramatically increased sensitivity to both DAG and tetracaine but a less obvious change in channel gating. These mutations introduced voltage dependence and gating kinetics without a change in apparent agonist affinity or efficacy. However, substitution of residue 204 with tryptophan resulted in more dramatic effects on gating as well as higher sensitivity to the two inhibitors. In fact, unlike the other mutants, the tryptophan mutant demonstrated a decrease in apparent agonist affinity and cAMP efficacy, in addition to the appearance of voltage-dependent gating. These results indicate that it is not merely the charge of the glutamate and aspartate residues that altered channel gating and inhibitor sensitivities; instead, the mutations may have changed the secondary structure or simply the flexibility of the loop region by introducing bulkier groups than the wild-type glycine. Alternatively, the replacement of glycine at position 204 may have changed the nature of interactions of this loop with other parts of the channel. The Hill coefficients from the DAG doseresponse curves of the more sensitive channels confirm that at least two or three molecules of DAG are typically required to inhibit a channel (discussed in more detail in the companion article
The Molf G204E, Rolf G204E, and Rolf G204D constructs provide the first demonstration of a point mutation that drastically alters inhibition by DAG or tetracaine without altering the apparent sensitivity to cyclic nucleotides. How could a change in the residue at position 204 have a similar dramatic effect on inhibition by two structurally different inhibitors without changing these gating parameters? Our results suggest that gating kinetics are more sensitive to changes in the allosteric conformational change than are standard equilibrium gating parameters such as apparent cGMP affinity and cAMP efficacy. Thus, unlike the wild-type olfactory channel, the aspartate and glutamate mutants demonstrated voltage-dependent gating kinetics, resembling those previously documented for the native rod CNG channel (
The voltage dependence was further investigated by plotting the doseresponse curves at +100 and -100 mV. As expected, a shift between these doseresponse curves was observed for the Brod and Rolf G204E channels, but not for the Rolf channel. These studies, demonstrating weakly voltage-dependent gating, are consistent with previous findings for Brod homomultimers (
and ß subunits), but not in the Brod
homomultimeric channels (
homomultimers is consistent with the studies by
Although the introduction of a negative charge at position 204 produced only subtle changes in gating properties, the substitution of a bulky hydrophobic group (tryptophan) had more severe consequences. Interestingly, this single point mutation converted the olfactory channel to one that behaves more like the rod channel, displaying lower apparent agonist affinity, cAMP efficacy, as well as more pronounced gating kinetics and voltage dependence. In light of the drastically altered gating properties of the Rolf G204W mutant, it is not surprising that its response to inhibitors is also like that of the rod channel. One possible explanation for the larger effect of the tryptophan molecule on channel gating is that its hydrophobic side chain may insert into the bilayer and disrupt the normal motions of the transmembrane segments that occur during allosteric transitions, in much the same way as we have proposed for the DAG molecule (see first model of Fig. 10 in companion article,
Since glycine is present at the equivalent of position 204 in both the rod and olfactory channels (Fig 1), other residue differences between the two channels must be responsible for the differential DAG inhibition of these two channels. In fact, a parallel study with chimeras of the two channels demonstrated that regions outside of the S2S3 loop of the rod channel can also convey DAG sensitivity (see companion article
In the lac permease of Escherichia coli, deletions in the loops were generally not disruptive to the protein's function unless they encroached on the boundaries with the transmembrane segments, where critical residues were proposed to actually be part of the transmembrane helix (
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Footnotes |
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1 Abbreviations used in this paper: Brod, bovine rod; CNG, cyclic nucleotidegated; DAG, diacylglycerol; Molf, mouse olfactory; Rolf, rat olfactory.
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Acknowledgements |
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We are grateful to 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 constructs. We also thank Elizabeth Seed, Wang Nguitragool, and Roberto Neisa for their technical assistance.
This work was supported by the American Heart Association of Rhode Island (grant No. 97-07721S) and the National Institutes of Health (grant No. EY07774).
Submitted: 10 May 2000
Revised: 22 September 2000
Accepted: 2 October 2000
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