Correspondence to: Roderick MacKinnon, Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, 1230 York Avenue, New York, NY 10021., mackinn{at}rockvax.rockefeller.edu (E-mail), Fax: 212-327-7289; (fax)
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Abstract |
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The pore of the catfish olfactory cyclic nucleotidegated (CNG) channel contains four conserved glutamate residues, one from each subunit, that form a high-affinity binding site for extracellular divalent cations. Previous work showed that these residues form two independent and equivalent high-pKa (~7.6) proton binding sites, giving rise to three pH-dependent conductance states, and it was suggested that the sites were formed by pairing of the glutamates into two independent carboxyl-carboxylates. To test further this physical picture, wild-type CNG subunits were coexpressed in Xenopus oocytes with subunits lacking the critical glutamate residue, and single channel currents through hybrid CNG channels containing one to three wild-type (WT) subunits were recorded. One of these hybrid channels had two pH-dependent conductance states whose occupancy was controlled by a single high-pKa protonation site. Expression of dimers of concatenated CNG channel subunits confirmed that this hybrid contained two WT and two mutant subunits, supporting the idea that a single protonation site is made from two glutamates (dimer expression also implied the subunit makeup of the other hybrid channels). Thus, the proton binding sites in the WT channel occur as a result of the pairing of two glutamate residues. This conclusion places these residues in close proximity to one another in the pore and implies that at any instant in time detailed fourfold symmetry is disrupted.
Key Words: ion channel permeation, proton block, Xenopus oocyte expression, ligand-gated ion channels, patch clamp
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Introduction |
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Changes in pH at the intracellular or extracellular face of an ion channel under physiological or laboratory-defined conditions can have strong effects on gating or permeation properties. pH-dependent ion channel behavior, controlled by the binding of protons to important functional regions of the channel protein, has been observed for a wide variety of channel types, including voltage-dependent Na+ (
In some cases, the effects of protons on ion permeation can provide insight into the detailed structure of the pore. One example is the promotion of subconductance states by the binding of H+ to the pore of the cardiac L-type voltage-dependent Ca2+ channel. Unitary Ca2+ channel recordings in cardiac ventricular myocytes showed that the binding of protons to a single extracellular site having a pKa of ~7.5 caused the channel to switch from a high-conductance state to a state with threefold lower conductance (
A similar phenomenon has been observed in the cloned catfish olfactory cyclic nucleotide-gated (CNG)1 channel, a nonselective cation channel having homology to voltage-gated potassium channels (
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The purpose of this study was to test the plausibility of the idea that the two protonation sites in the pore of the catfish olfactory CNG channel consist of two physically separate carboxyl-carboxylate pairs. The strategy we used was to create hybrid CNG channels containing a mixture of wild-type (WT) and Glu333Gly (E333G) subunits. If the sites are separate carboxyl-carboxylates, we reasoned, it should be possible to isolate a hybrid channel containing one, but not both, of the original protonation sites intact.
By expressing a mixture of WT and E333G subunits, we formed functionally WT channels, pure mutant channels, and four novel channel types, a result consistent with the postulated tetrameric structure of CNG channels. One of the novel channels, which we named Type B, had two pH-dependent conductance states whose occupancy was governed by protonation at a single site having a pKa of 6.8. Expression of tandem dimer constructs enabled us to determine the number of WT and E333G subunits in all of the hybrid channels and revealed that the Type B channel contained two WT and two E333G subunits. We conclude that we were able to isolate, in the Type B hybrid channel, a single protonation site formed by two glutamate carboxyl groups with properties very similar to those in the native channel. Our results corroborate the hypothesis that the protonation sites in the native channel are structurally independent carboxyl-carboxylates.
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Materials and Methods |
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Molecular Biology
The catfish olfactory CNG channel subunit was carried in the pGEMHE plasmid (construct kindly provided by E. Goulding and S. Siegelbaum of Columbia University, New York;
Dimer constructs were made as follows. The catfish olfactory CNG channel subunit (WT or E333G) was subcloned into the pRSET plasmid (BamHI to HindIII) just 3' to a 102-bp sequence (NcoI to BamHI) encoding a 34 amino acid peptide linker. The linker, originally designed for antibody studies of the channel, contained a polyhistidine stretch followed by a T7 epitope tag and an aspartate-rich sequence. The entire linker plus channel construct (NcoI to HindIII) was excised from pRSET and inserted into a pGEMHE-CNG channel construct that had been mutated to give an NcoI site at the 3' end of the
subunit coding sequence. The result was a construct in pGEMHE (BamHI to HindIII) consisting of two complete catfish olfactory CNG channel
subunits separated by the 102-bp linker sequence. To reduce the likelihood of contamination of dimer DNA or RNA by monomers, the following measures were taken: (a) only recA- strains of Escherichia coli were used to carry the dimer plasmids; (b) after the dimer ligation reaction, single colonies were picked and shown to contain a single dimer-sized species by agarose gel electrophoresis; and (c) after in vitro RNA synthesis, dimer RNA was compared side-by-side with monomer RNA on an agarose gel and shown to be free from contamination by the monomer band, which ran at a distinct position. Dimer constructs were made from the WT and E333G subunits in all possible combinations: WT:WT, WT:E333G, E333G:WT, and E333G:E333G. Dimer RNA was synthesized using T7 RNA polymerase after linearization with SphI.
Electrophysiology
Xenopus laevis oocytes were prepared and injected with CNG channel RNA as previously described (. Single-channel currents were recorded 13 d after RNA injection using an Axopatch 200 amplifier (Axon Instruments). The amplifier output was filtered at 2 kHz and sampled at 10 kHz using a DAP data collection board (Microstar Laboratories). All-points amplitude histograms were constructed off-line using an analysis program written in Microsoft QuickBASIC. The bin-width of the histograms was 0.05 pA, and at least 500,000 sample points (50 s) of data were used to construct each histogram. Histograms were constructed primarily from open-channel activity, with a small amount of baseline activity included as a reference.
The internal and external solutions were made using 2H2O (deuterium oxide; Sigma Chemical CO.) to slow the kinetics of protonation and deprotonation events, as previously described (
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Results |
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Injection of a Mixture of WT and Glu333Gly CNG Subunits Gives Rise to WT Channels, Pure Mutant Channels, and Four Hybrid CNG Channel Types
Our primary objective was to test the independence of the two protonation sites found in the WT catfish olfactory CNG channel. To do this, we asked whether it was possible to use a mixture of WT and E333G subunits to create a hybrid channel containing one but not both sites. A 2:1 mixture of WT and E333G subunit RNA was injected into Xenopus oocytes, and single CNG channels were recorded from inside-out patches at a holding voltage of -80 mV, with 130 mM NaCl, pH 7.6, on both sides of the membrane. The varieties of single channels found are shown in Figure 2 A. In the first two columns, individual examples of current traces are presented alongside amplitude histograms calculated for the same channels. Pure mutant channels with a single conductance state of ~25 pS were observed (Figure 2 A, top), as were WT channels having the usual three conductance states of 6570, 3540, and 1520 pS (Figure 2 A, bottom). In addition, four novel types of hybrid channels were found, which we have called Types A, B, C, and D. The Type A channel had no single well-defined conductance state, spending most of its time at low (~30 pS) conductances, but also displaying brief spikes to higher conductances (which gave rise to the long tail in the amplitude distribution). The Type B channel appeared to have two well-separated conductance states of ~5065 and ~2540 pS, with the higher conductance state favored at pH 7.6. The Type C channel appeared to jump rapidly between poorly distinguished conductance levels and visited both lower and higher conductances from its main conductance level of ~5060 pS, behavior that gave both high- and low-conductance tails in the amplitude histogram. The Type D channel appeared in single-channel records to show behavior similar to that of the WT channel, with transitions among three conductance states. Its amplitude histogram, however, always showed only two recognizable peaks (~7075 and ~2530 pS), perhaps because the lowest-conductance state was so infrequently and briefly occupied at pH 7.6. The third column shows average amplitude histograms calculated from all of the individual histograms assigned to each category (pure mutant, A, B, C, D, or WT). The shapes of the group average histograms were similar to those of the individual examples, underscoring the uniqueness of the hybrid channel types. The broader peaks seen in the group average Type B and WT histograms reflect the variation we observed in the absolute amplitude (although not in the shape) of these channel types. Figure 2 B shows the number of each channel type found, out of a total of 65 single-channel patches pulled from WT and E333G coinjected oocytes. As expected, the least common species were the homomultimeric WT channels (two observed) and pure mutant channels (six observed). Of the hybrid channels, Types A and B were found more than twice as frequently as Types C and D.
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Figure 3 shows amplitude histograms compiled from examples of the four hybrid channel types at a range of extracellular pH values. Recordings were made from outside-out patches, and pH changes were made by moving the patches between microcapillary perfusion tubes. While the behavior of Type A channels did not change significantly between pH 8.5 and 6.0 (aside from a slight flattening of the high-conductance tail of the amplitude histogram), the behavior of Types B, C, and D was strongly pH dependent. Type B channels appeared to undergo a smooth transition from a well-defined high-conductance state to a well-defined low-conductance state as the pH was lowered. Type C channels also shifted to lower conductances at lower pH values, although it was not possible to discern two clearly separable conductance states. For the Type D channel, a transition between two well-defined conductance states at high pH was followed by a more gradual shift to lower conductances at low pH. While the amplitude distributions of Type B channels kept their distinctive two-peaked shape even at pH 6.0, the Type A, C, and D distributions all seemed to converge at low pH on a common shape having a low-conductance hump with a long, high-conductance tail.
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The pH Dependence of the "Type B" Hybrid CNG Channel Is Consistent with Protonation at a Single Site
Of the four hybrid channels, the Type B channel, exhibiting two distinct conductance states over a wide pH range, seemed like the best candidate for a hybrid having one of the WT protonation sites. Amplitude histograms were compiled from Type B channel activity in outside-out patches at various values of extracellular pH and fitted to a sum of three Gaussian functions corresponding to the closed state and two open states of the channel. Figure 4 A shows amplitude histograms from the same channel at three pH values; the top (pH 8.0) histogram appears with its three-Gaussian fit (solid line). The relative area under the Gaussian functions for the nonzero conductance states, a measure of the relative occupancy of the states, was calculated for three channels and plotted as a function of pH (Figure 4 B). The dotted lines in Figure 4 B show the predictions of a model in which transitions from the high to the low state are governed by protonation at a single site (see Figure 4, legend). The good agreement with the data indicates that the steepness of the transition between states in the Type B channel is consistent with a single-site mechanism. The best fit to the data gives a pKa for the single site of 6.8.
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The Types B and C Hybrid Channels Contain Two WT and Two E333G Subunits
To gain a better understanding of the subunit composition of the hybrid CNG channels, we constructed and expressed tandem dimers of WT and E333G subunits. Our hypothesis was that the subunit composition of channels formed from dimers, instead of coexpressed monomers, would be more tightly constrained. In the optimal case, in which dimers associate in one orientation to form full tetrameric channels, one would expect expression of each single type of dimer to give rise to a single type of channel. The dimers used in these experiments consisted of two complete CNG subunits joined by a 34 amino acid linker (see MATERIALS AND METHODS).
Figure 5 shows the behavior of channels observed in inside-out patches after several types of dimers were expressed. Expression of the dimer consisting of a WT subunit linked in tandem to an E333G subunit (the E:G dimer, named after the residues at position 333 in each subunit) gave rise to two and only two of the hybrid channel types, Types B and C. These were identified on the basis of the shapes of their amplitude histograms at pH 7.6 and their pH dependence in outside-out patches (data not shown). Expression of the opposite dimer, G:E, gave the same two types of hybrid channels, as did expression of a 2:1 mixture of the E:E and G:G dimers (which also gave WT and pure mutant channels, not shown). The fact that expression of the E:G or G:E dimer alone produced more than one type of channelas well as the fact that coexpression of the E:E and G:G dimers produced two different hybrid channelssuggested that the dimers were associating in more than one way.
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One explanation for the data of Figure 5 is that the CNG dimers can assemble into tetrameric channels in all three possible dimerdimer configurations, illustrated in Figure 6, top. In the first configuration (i), dimers associate "head-to-tail," with the A protomer of one dimer contacting the B protomer of the other. In the second configuration (ii), dimers associate "head-to-head," with the A and B protomers of the two dimers contacting each other. In the third configuration (iii), the dimers interlock to form channels, and the A and B protomers of each dimer sit at opposite corners of the channel. This scheme depends on the assumptions that CNG channels are fourfold symmetric tetramers and that all of the dimers associate as dimers, contributing both of their subunits to each of the channel species formed.
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As shown in Figure 6, bottom, the first two configurations (i and ii) are sufficient to give two channel structures when the E:G or G:E dimer is expressed alone. These structures are each predicted to have two WT and two E333G subunits, with the WT subunits lying across the channel from each other (i) or adjacent to each other along a side of the channel (ii). When the E:E and G:G dimers are coexpressed, association in the first two configurations would give rise to only one of these two structures (the structure having adjacent WT subunits), and it becomes necessary to invoke the third configuration (iii) to produce the structure having WT subunits at opposite corners of the channel. As indicated in the figure, coinjection of the E:E and G:G dimers would also be expected to give rise to WT and pure mutant channels.
If the scheme shown in Figure 6 is correct, then the Type B and Type C channels each have two WT and two E333G subunits, with one of them having adjacent and the other opposite WT subunits; the data in Figure 5 do not indicate with certainty which channel type corresponds to which arrangement. Regardless of their orientation, however, the presence of exactly two WT subunits, and hence two pore glutamates, in the Type B channel is a further indication that this channel could contain a single carboxyl-carboxylate that is similar to those formed in the pore of the WT channel.
Identities of the Type A and Type D Channels
Figure 7 shows the types of channels observed in inside-out patches when the E:G dimer was coexpressed with either the G:G (top) or the E:E (bottom) dimer. In both cases, coexpression gave rise to the channels expected from expression of the dimers individually: pure mutant, Type B, and Type C channels for the coexpression of the E:G and G:G dimers; WT, Type B, and Type C channels for the coexpression of the E:G and E:E dimers (the pure mutant and WT channels are not shown in the figure). In both cases, an additional hybrid channel type was also found. For the E:G + G:G coexpression, this additional hybrid channel was the Type A channel, while for the E:G + E:E coexpression, the additional channel was the Type D channel. As before, the hybrid channels were identified based on the shapes of their amplitude histograms at pH 7.6 and their pH dependence in outside-out patches (not shown).
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The additional hybrid channel produced in each case, not expected from expression of the dimers individually, most likely arose from the two coexpressed dimers coming together to form a unique channel. This implies that the Type A channel contained one WT subunit and three E333G subunits, while the Type D channel contained three WT subunits and one E333G subunit. Only one unique hybrid is expected in each case, since coassociation of the coexpressed dimers in all three of the configurations shown in Figure 6, top, would give rise to the same subunit arrangement. Types A and D channels were never seen when the E:G, G:E, or E:E + G:G dimers were expressed (Figure 5), consistent with the conclusion that they have a one WT, three mutant or three WT, one mutant subunit arrangement.
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Discussion |
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The Nature of the Protonation Sites in the Catfish Olfactory CNG Channel
The central goal of this study was to test the idea, proposed by
The present results, taken together with the earlier results of
Subunit Stoichiometry of the Catfish Olfactory CNG Channel
Given the homology between the channel under study and potassium channels, which have been shown to have a stoichiometry of four subunits per channel (
Our conclusion closely parallels the findings of
Molecular Identities of the Hybrid CNG Channels
Expression of CNG channel dimers made it possible to draw conclusions about the subunit makeup of the four types of hybrid channels we observed when WT and E333G monomers were coexpressed. Figure 8 shows the possible WT and E333G subunit combinations expected for a tetrameric channel and correlates these combinations with the hybrid channels that were observed. The Type A channel could be unambiguously assigned to a specific structure since coexpression of the E:G + G:G dimer combination was not sensitive to the variability of dimer association. Since the Type A channel arose uniquely when the E:G and G:G dimers were coexpressed, we conclude that this channel has only one WT subunit and therefore only one pore glutamate. This is consistent with this channel's lack of a strong pH dependence between pH 8.5 and 6.0, since one would expect a lone carboxyl group to have a pKa several units lower.
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The Type B and Type C channels each contain two WT and two E333G subunits, although the flexibility of dimer association prevented us from determining which of these had two adjacent WT subunits and which had two opposite WT subunits. This leaves doubt as to the precise configuration of the carboxyl-carboxylate interaction in the Type B channelit is possible that it occurs across the channel between opposite subunits or along a side of the channel between adjacent subunits. Which of these possibilities is more plausible depends critically on the position and angle at which Glu333 projects into the channel pore. The fact that the Type C channel does show pH dependence between pH 8.5 and 6 presents the possibility that its two glutamates can combine to form a protonation site of fairly high pKa. Figure 3 suggests that in this channel, protonation at a site with a pKa of ~6.25 causes a gradual shift to a lower conductance state of ~2025 pS. The Type C channel resembles the EIIQ L-type Ca2+ channel mutation studied by
The Type D channel, which arose uniquely when the E:G and E:E dimers were coexpressed, could be unambiguously assigned to a structure having three WT subunits (and hence three pore glutamates). Consequently, its conductance shows strong pH dependence. Since this channel has glutamates positioned next to each other and across the channel from each other, it should accept protons both like a Type B and like a Type C channel. This is consistent with the pH dependence shown in Figure 3, which shows a clear Type Blike transition between two conductance states at high pH followed by a less well-resolved Type Clike transition to a lower conductance state at lower pH.
The WT channel, containing all four glutamates, would also be expected to show both Type B and Type C behavior. Protonation in the Type B mode (i.e., via independent carboxyl-carboxylate interactions) would give the transitions among three clear conductance states that are the hallmark of this channel. Protonation in the Type C mode, causing a poorly resolved transition to an intermediate conductance state, might be expected to affect the behavior of the WT channel more subtly.
We modeled this effect by adding a fourth conductance state to the channel, as depicted in Figure 9 A. In the model, Type B protonation at the two independent and equivalent carboxyl-carboxylates causes transitions along the top row of states, from the high-conductance state s1 (6570 pS) to the middle- and low-conductance states, s2 (4045 pS) and s3 (1520 pS) (these are the same transitions diagrammed in Figure 1 C). Type C protonation shifts the channel to an intermediate state s4 whose conductance, estimating from Figure 3, is ~2025 pS, between the conductances of s2 and s3, an estimate that depends on the assumption that protonation in the Type C mode has the same effect on the WT channel, which has four pore glutamates, as on the Type C channel, which has only two glutamates. In theory, a second Type C protonation is possible in the WT channel, which would be expected to give rise to a fifth conductance state in which both Type C sites are occupied. However, since we could only measure one Type C transition in the Type C channel (which contains only two glutamates), and since we have no way of predicting the pH range of the second Type C protonation or its effect on the channel conductance, we limit our model to only one Type C transition. This model is therefore incomplete and should be regarded as a first approximation.
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Because Type C, like Type B, protonation should occur in the WT channel at either of two sites and is assumed to be diffusion limited, we take the transition rate from s1 to s2 or s4 to equal 2[H+], where
= 6.4 x 109 M-1*s-1 (following
+ at 1,920 s-1 and
- at 200 s-1.
Figure 9 B compares a WT amplitude histogram recorded at pH 7.6 and -80 mV with amplitude histograms generated from the model under the same conditions and suggests that Type C protonation in the WT channel might explain a subtle but consistent feature of WT behavior. As indicated by the arrow in Figure 9 B, the WT channel consistently tends to visit current levels between the low- and middle-conductance states more often than expected; the extra density in this region is greater than can be explained by overlap of the Gaussian functions for the two neighboring states. This feature was observed in every WT channel recorded in this study (see Figure 1 and Figure 2) and is evident in the original data of
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Acknowledgements |
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We thank Michael J. Root and Chul-Seung Park for helpful discussions.
This work was supported by National Institutes of Health grant GM47400 and by a Harvard Medical School Department of Neurobiology Quan Predoctoral Fellowship (to J.A. Morrill). R. MacKinnon is an Investigator in the Howard Hughes Medical Institute.
Submitted: March 15, 1999; Revised: May 18, 1999; Accepted: May 19, 1999.
1used in this paper: CNG, cyclic nucleotidegated; E333G, Glu333Gly; WT, wild type
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