Correspondence to Sharona E. Gordon: seg{at}u.washington.edu
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ABSTRACT |
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Key Words: CNG channel gating oxidation structure cGMP
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INTRODUCTION |
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Although both the cyclic nucleotide-binding domain and the ion-conducting pore in CNG channels have been structurally defined, based on their homology to ion channels whose structural elements have been solved, the mechanism by which the energy provided by cyclic nucleotide binding is converted into opening of the pore is not understood. A region linking the last membrane-spanning region (S6) to the cyclic nucleotide binding domain in the COOH terminus (the C-linker) has been shown to play an important role in channel gating. Previous experiments suggest that the C-linker controls coupling of ligand binding to channel gating, and that the movement of this region is closely coupled to channel activation (Gordon and Zagotta, 1995a,c
; Tibbs et al., 1997
; Paoletti et al., 1999
; Johnson and Zagotta, 2001
).
Divalent transitional metal (Ni2+) binding to the C-linker of CNG channels has been used to demonstrate that open-state specific intersubunit interactions occur between proximal C-linker regions (Gordon and Zagotta, 1995a,b
; Johnson and Zagotta, 2001
). Given that amino acids coordinating Ni2+ must be within
4 Å of each other (Maroney, 1999
), these experiments strongly suggest that at least part of the proximal C-linker region must be a site for intersubunit proximity. Furthermore, at several sites, Ni2+ coordination occurs preferentially in the open state, providing a constraint on the proximity of this region in the open state. A later study using a histidine scan of the proximal C-linker of CNGA1 reported that stripes of sites separated by 50 degrees on an
-helix produced Ni2+ potentiation or Ni2+ inhibition (Johnson and Zagotta, 2001
). These results suggest that the C-linker region undergoes a rotational movement during channel activation. This rotation may initiate movement of S6 and pore opening.
Hyperpolarization-activated, cyclic nucleotide-modulated channels (HCN) contain a C-linker region that is closely related to CNGA1 based on sequence similarity. The sequences of HCN2 and CNGA1 in the C-linker are 22% identical and 45% conserved overall (Fig. 1 A). Recently, a crystal structure of the COOH-terminal region of the HCN2 channel was solved to 2.0 Å resolution (Zagotta et al., 2003). This structure includes the C-linker and the cyclic nucleotide-binding domain (CNBD) bound to cAMP. In this structure, four HCN2 COOH termini are associated to form a tetramer. The C-linker region consists of six
-helices, designated A' to F', which are separated by short loops (Fig. 1 A). The C-linker regions also form a large interface of subunitsubunit interaction with 2,300 Å2 of buried solvent-accessible surface area. In contrast, the CNBD regions show very little intersubunit contact. This structure suggests that the C-linker regions mediate the primary intersubunit interactions of the COOH termini of HCN2. Based on the high sequence similarity between the COOH termini of HCN2 and CNGA1 (Fig. 1 A), the CNGA1 C-linker regions may have similar structural and functional importance.
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In this study, we explored the intersubunit proximity between the C-linker regions of CNGA1 in functional channels using a site-specific cysteine substitution method (Falke et al., 1988). We found that intersubunit disulfide bonds can be formed between the A' helices of the C-linker regions during channel activation, indicating that these regions are in close proximity. Under our experimental conditions, most of the intersubunit disulfide bonds in this region potentiated channel activation, reflected as an increased apparent affinity for cGMP. We mapped the residues that formed intersubunit disulfide bonds onto a homology model of CNGA1, based on the structure of HCN2. Our data are not compatible with a model in which the C-linker conformation represents that of the open state, and suggest that the conformation seen in the structure may in fact represent the closed state.
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MATERIALS AND METHODS |
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Heterologous Expression of Channels in Xenopus Oocytes
Segments of ovary were removed from anesthetized Xenopus laevis. After gross mechanical isolation, oocytes were isolated by agitating the sections of ovary in a Ca2+-free collagenase (2.3 mg/ml) solution for up to 1.5 h. The cells were then rinsed and stored in frog Ringers solution at 14°C. Oocytes were generally injected with 40 nl cRNA solutions (1 µg/µl) within 2 d of harvest. Electrophysiological recordings were performed 210 d after injection.
Electrophysiology
Inside-out membrane patches were obtained by using fire polished borosilicate pipettes (1.5 mM OD, 1.2 mM ID; Sutter Instrument Co.). Recordings were made using symmetrical NaCl/HEPES/EDTA solutions consisting of 130 mM NaCl, 3 mM HEPES, 0.2 mM EDTA (pH 7.2) with cGMP or cAMP added to the intracellular solution only. Copper (II) phenanthroline (Cu/P) (Kobashi, 1968) was prepared as follows: a 5 mM 1,10-phenanthroline stock in dry ethanol and a 1.5 mM cupric sulfate stock in water were diluted to final concentrations of 5 µM phenanthroline and 1.5 µM cupric sulfate in a solution containing 130 mM NaCl and 3 mM HEPES (pH 7.2); 2 mM cGMP or 128 µM cGMP (as indicated in the text) were present in this solution as well. Cu/P solutions were used for up to 2 d after preparation. Cu/P was applied to the intracellular side of patches for 310 min, as indicated in the text and figure legends. Dithiothereitol (DTT) was made as a 1 M stock in the NaCl/HEPES/EDTA solution and diluted to a final concentration of 5 mM in the NaCl/HEPES/EDTA solution together with 64 µM cGMP. The DTT solution was made fresh daily.
For macroscopic current measurements, pipettes were polished to a resistance between 0.3 and 1 M. Currents were low-pass filtered at 2 kHz and sampled at 10 kHz with an Axopatch 200B (Axon Instruments, Inc.). All recordings were made at room temperature. Data were acquired and analyzed with PULSE data acquisition software (Heka Elektronik) and were plotted and fitted using Igor Pro (Wavemetrics Inc.). All currents shown have had currents in the absence of cyclic nucleotide subtracted. All currents were measured at +100 mV. For each experiment, currents at different cGMP concentrations were measured, and we waited until the currents had stabilized before proceeding. This delay was necessary because of the "run-up" caused by dephosphorylation of the channel by endogenous patch-associated phosphatases (Gordon et al., 1992
; Molokanova et al., 1997
). Smooth curves shown in doseresponse relations are fits of the data to the Hill equation: I = Imax ([cGMP]n/(K1/2n + [cGMP]n)). Data are reported as the mean ± SEM. Statistical significance was assayed with two-tailed Student's t tests.
For single channel recordings, pipettes were polished to a resistance between 8 and 12 M. Currents were low-pass filtered at 5 kHz (eight-pole Bessel) and sampled at 10 kHz with an EPC10 amplifier (Heka Elektronik). Patches were held at 0 mV and stepped to +50 mV for 1 s. The presence of single channels in the patches was confirmed by application of 2 mM cGMP. Open probability (Po) was calculated from fits to the data with two Gaussians. The area under the curve representing open channels relative to the total area was taken as Po. Cu/P was applied to the intracellular side of the patches for 10 min for all single-channel experiments. Other solutions and software for data acquisition and analysis were the same as for the macroscopic current measurements.
Online Supplemental Material
Fig. S1 shows that Ni2+ both potentiates and inhibits 417H. cGMP doseresponse curves without Ni2+ (open circles) and with 1 µM Ni2+ (filled circles). The smooth curves represent fits with the Hill equation with K1/2 = 32.3 µM, Hill slope = 1.9 initially, and K1/2 = 22 µM, Hill slope = 1.1 with 1 µM Ni2+. The mean K1/2 for activation by cGMP decreased from 32 ± 5.7 µM in the absence of Ni2+ to 17.4 ± 3.5 µM in the presence of Ni2+ (n = 3). The supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.200409187/DC1.
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RESULTS |
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We substituted a cysteine for each of the amino acids from 417 to 424 individually in the CNGA1cysless (Matulef et al., 1999) background. All the mutants were functional except 421C. Mutant channels were expressed in Xenopus oocytes and examined using the inside-out configuration of the patch-clamp technique. To ensure that introduction of each cysteine did not prevent formation of functional, cyclic nucleotide-activated channels, we first measured the doseresponse relation for activation of the mutant channels by cGMP (Fig. 2, open circles) and calculated the K1/2 (Table I, K1/2 initial; see MATERIALS AND METHODS), and also measured the fractional activation by a saturating concentration of the partial agonist cAMP (Fig. 3, open circles). We next used Cu/P to induce disulfide bond formation in each of the cysteine mutants. Two general types of outcomes are possible. If the regions containing the cysteines move during gating, then a disulfide bond that locks two of these regions together would be expected to perturb gating. If relative movement of the region containing the cysteines does not occur as part of gating, then locking the regions together through disulfide bond formation is not expected to perturb function. A third possibility, that no disulfide bond forms, cannot be functionally distinguished from a scenario in which a disulfide bond forms but does not alter channel properties.
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Cu/P Effect Can Be Reversed by DTT
Under our oxidation conditions, Cu/P is expected to induce formation of a disulfide bond between cysteines from two subunits. However, it is possible that Cu/P would overoxidize the cysteines into sulfinic, sulfenic, or sulfonic acid (Torchinskii, 1974). One way to differentiate between such overoxidation and disulfide bond formation is to use the reducing agent DTT, which will reduce disulfide bonds but not overoxidized cysteines (Torchinskii, 1974
). Fig. 4 shows examples of experiments with 417C, in which DTT was applied after Cu/P treatment. For 417C, the cGMP doseresponse curve was measured initially, after Cu/P treatment, and after DTT treatment (Fig. 4 A, open, black, and red, respectively). Cu/P potentiated 417C function, shifting the cGMP doseresponse curve to the left. Subsequent application of 5 mM DTT for 10 min to the intracellular side of the patch substantially reversed the Cu/P effect, shifting the doseresponse relation for activation by cGMP back to the right.
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Potentiation and Inhibition by Cu/P
We next asked whether the combination of potentiation and inhibition observed at position 418 was an artifact of disulfide bond formation. We wondered if a mixture of potentiation and inhibition could be observed with Ni2+ as well. When we applied 1 µM Ni2+ to 417H channels, we observed both a reduction in the current activated by high concentrations of cGMP and an increase in the current activated by quite low concentrations of cGMP (Fig. S1, available at http://www.jgp.org/cgi/content/full/jgp.200409187/DC1), in contrast to previous reports (Gordon and Zagotta, 1995b; Johnson and Zagotta, 2001
). This finding suggests that the dual effect of potentiation and inhibition is not unique to Cu/P modulation, but can be observed with Ni2+ binding as well.
In our experiments, Cu/P treatment of 418C potentiated gating, as measured by low concentrations of cGMP and by the fractional activation by cAMP, but also decreased the maximal current activated by cGMP. Does disulfide bond formation at 418C produce potentiation or inhibition? This is an important point, because we interpret potentiation as open-state specific proximity and inhibition as closed-state specific proximity, two mutually exclusive possibilities.
There are two general ways in which both potentiation and inhibition could be observed. Both potentiation and inhibition could be present in every channel, with each channel behaving in the same way. Alternatively, Cu/P could produce two populations of channels, each showing only potentiation or only inhibition. One simple way for each channel to show both inhibition and potentiation is if an intersubunit disulfide bond between 418C subunits causes a decrease in channel conductance (the decrease in the maximal current), but an increase in channel open probability (the potentiation at subsaturating cGMP). The proximal C-linker region is at the end of S6, a likely location of the channel gate (Flynn and Zagotta, 2003). It is conceivable that a conformational change in this region induced by disulfide bond formation could cause changes in both ion permeation and in gating. The second general way to produce potentiation and inhibition is if two populations of channels are present, one with a decreased open probability, and the other with an increased open probability. We ought to be able to distinguish between these two models using single-channel recording.
We recorded the activity of single 418C and CNGA1cysless (as a control) channels before and after treatment with Cu/P (Fig. 5, left), and plotted all-points histograms to determine their open probability (Fig. 5, right). In each experiment, Cu/P was applied for 10 min, and currents were recorded at +50 mV with a low cGMP concentration (as indicated in the figure legend). The single channel conductance was 26 pS for both channels before Cu/P application, which was in the same range as that of the wild-type CNGA1 channel (Sunderman and Zagotta, 1999). Interestingly, we found that Cu/P treatment produced two populations of channels in 418C, one potentiated and the other inhibited. Among the seven patches tested, three of them showed an increase in open probability (Po) at 4 µM cGMP after Cu/P application (Fig. 5 A, Po = 0.09 ± 0.012 initially, Po=0.83 ± 0.087 after Cu/P). For a saturating concentration of cGMP (2 mM), the Po in these patches was initially 0.91 ± 0.054 and was 0.90 ± 0.066 after Cu/P treatment (unpublished data). The single channel conductance was not altered by Cu/P treatment. This indicates Cu/P potentiated 418C through an effect on channel gating. For the other four patches, Po was 0.08 ±0.001 initially, and was reduced to essentially zero after Cu/P treatment both at 4 µM (Fig. 5 B) and 2 mM cGMP (not depicted). In all four of these inhibited patches, we recorded a total of <10 opening events after Cu/P treatment. This rate of opening was not sufficient to calculate Po, but is interesting because those few openings that were observed had the same unitary conductance as observed before Cu/P treatment (unpublished data). The inhibition of this population of channels correlates with the inhibition of current produced by Cu/P treatment at the macroscopic level. As a control, we also measured the single-channel effects of Cu/P treatment on CNGA1cysless channels. We did not find a significant effect of Cu/P on the open probability or the single-channel conductance (Fig. 5 C; at 2 µM cGMP, Po = 0.03 ± 0.014 initially, Po = 0.09 ± 0.026 after Cu/P, n = 2, single channel conductance = 26 pS).
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DISCUSSION |
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In the homology model of the C-linker of CNGA1 shown in Fig. 1 B, the residues at the 420 position of both adjacent and opposite subunits are much too far apart to either form a Ni2+ coordination site (420H) or to form a disulfide bond (420C). Our data now show that the functional proximity between subunits is observed not only at the 420 position, but at 417 and 418 as well. Furthermore, in all cases, formation of a disulfide bond between cysteines at a given site produced potentiation. Potentiation of gating indicates an energetic stabilization of the open conformation of the channel relative to the closed conformation. Another way to state this is that the channels require less energy to open when the disulfide bond is formed compared with when the cysteines are reduced and the A' helices are free to move. We infer from this potentiation that the conformation of the channel trapped by the disulfide bond most closely resembles the open conformation.
Our data are incompatible with a model in which 417, 418, and 420 in different subunits are tens of Angstroms apart, as depicted in Fig. 1 B. We are led, then, to propose the following model for intersubunit interactions in the C-linkers of both CNG and HCN channels. Our hypothesis is depicted in Fig. 7. This cartoon shows the C-linker region represented in both the closed and open state. The closed state (Fig. 7 A) is identical to that of the HCN2 crystal structure. Although cAMP is bound to the CNBD in the HCN2 crystal structure, and the CNBD structure likely represents that of the open configuration, we believe that the C-linker in the structure is in the closed conformation. For the open state (Fig. 7 B), we have rotated the A' and B' helices as a unit, by introducing a rotation around the B'-C' loop. This rotation brings the A' helices nearly perpendicular to the membrane, as in the Johnson and Zagotta model (Johnson and Zagotta, 2001). We moved both the A' and B' helices as a unit because movement around the A'-B' loop alone could not bring the 417420 positions close enough to form a disulfide bond. However, the required proximity between the A' helices does not uniquely constrain the B' helices or other regions of the model. In functional channels, binding of cyclic nucleotide to the CNBD (not depicted) would cause a conformational change from the closed state, with A' helices at a distance, to the open state, with A' helices in proximity. This conformational change would be directly coupled to opening of the ion-conducting pore.
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What is the source of the two types of intersubunit interactions observed in 418C? Could one population arise from disulfide bond formation between subunits of different channels? Our observation of two populations at the single-channel level indicates this is not the case. We present two different ways different populations could arise in Fig. 6. In one model, disulfide bonds could form between either adjacent or opposite subunits, each with a different functional effect (Fig. 6 A). In the second model, formation of one disulfide bond would produce one effect, and formation of the second would produce the opposite functional effect (Fig. 6 B). We observed that the current activated by 2 µM cGMP was first potentiated by Cu/P, and was then inhibited after longer cumulative exposure to Cu/P. Although this time dependence does not rule out the model in which the two functional populations are based on disulfide bonds with two different orientations, it would seem that time dependence is more compatible with the model in which the two functional populations are based on having either one or two disulfide bonds. The potentiated population, observed with short time Cu/P treatments, would arise from formation of a single disulfide bond. The inhibited population would then arise later, as the second disulfide bond formed.
Inhibition of CNGA2 channels with Ni2+ has also been reported (Gordon and Zagotta, 1995b). Wild-type CNGA2 channels have a histidine at the position corresponding to 417, and a glutamine at the position corresponding to 420. Treatment of wild-type CNGA2 channels with Ni2+ shifts the doseresponse relation for cGMP to the right, increasing the K1/2 from 2.3 to 6 µM (Gordon and Zagotta, 1995b
). The inhibition of CNGA2 channels by Ni2+ is unambiguous. We propose that this inhibition arises from the same mechanism that produced inhibition in CNGA1-417H. However, subtle differences in structure between CNGA1 and CNGA2 might prevent the relatively small potentiation produced by Ni2+ in CNGA1-417H from occurring in CNGA2. This is most likely to occur if 417 is at a position that undergoes less movement during channel gating, relative to 420. Our model in Fig. 7 depicts 417 as near the fulcrum for movement, with proximity between subunits in both open and closed channels.
In summary, we have used trapping of the A' helices through disulfide bond formation to ask whether the crystal structure of the C-linker of HCN2 represents an open or closed conformation in CNG channel. Because each subunit contains only a single cysteine, any disulfide bond formation must occur between the same cysteine on different subunits. Three positions, 417, 418, and 420, formed disulfide bonds that potentiated activation of the channels. We interpret this potentiation to mean that the disulfide bond trapped the channels in a conformation more similar to the open state than to the closed state. These data are not compatible with a model in which the C-linker of the HCN2 structure represents the open channel conformation. Although cAMP is bound to the CNBD in the crystal structure and the CNBD observed in the structure almost certainly represents the open-channel conformation, our results indicate that the C-linker may be in the closed-channel conformation. Furthermore, the open-state specific proximity between subunits in the 417420 region indicates that the A' helices form tilted bundles perpendicular to the axis of the membrane in open channels. Channel closure may then involve a relaxation of the A' helices to the position parallel to the axis of the membrane, as observed in the crystal structure of HCN2.
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ACKNOWLEDGMENTS |
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This work was funded by a generous grant from the National Eye Institute (R01 EY013007 to S.E. Gordon).
David C. Gadsby served as editor.
Submitted: 28 September 2004
Accepted: 24 January 2005
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REFERENCES |
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