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* Department of Physiology and Biophysics, The Chicago Medical School, North Chicago, Illinois 60064; and Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021
Correspondence: Address reprint requests to Dr. Henry Sackin, Dept. of Physiology and Biophysics, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Rd., North Chicago, IL 60064. Tel.: 847-578-8329; Fax: 847-578-3265; E-mail: henry.sackin{at}rosalindfranklin.edu.
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ABSTRACT |
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INTRODUCTION |
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–4), and inward rectifiers in particular (5,6), the mechanism of K channel gating is still unresolved. In the Kir1.1 inward rectifier family there is evidence that the pH gate appears to be centered at a convergence of four hydrophobic residues at the cytoplasmic apex of the inner transmembrane (TM) helices (5–7). Conceivably, a gate located at this position could open and close by a hinging mechanism (5,8–10) or by a rotation of adjacent subunits, or both (6).
Two glycine residues on the inner (M2) transmembrane helices are highly conserved throughout the inward rectifier family (Fig. 1, sequence alignment). The more N-terminal of these glycines (G148-Kir1.1b) has been implicated as a hinge point for bending of the inner helix during opening and closing of inward rectifiers (6,10), as well as in KcsA and MthK (9) and Shaker (8).
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,10). In the Shaker Kv channel, the residue homologous to G157-Kir1.1b is contained within the amino acid sequence: Pro-Val-Pro, which may function as the physical gate of the Shaker channel (1,11–13). In the present study, we examine whether these conserved glycines: G148 and G157 are involved in the pH gating mechanism of the renal inward rectifier, Kir1.1b (ROMK). Our results indicate that pH gating can still occur when one or both glycines are replaced by less flexible alanines. However, this shifts the apparent pKa into the alkaline range, making these mutant channels much harder to open relative to wild-type ROMK.
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METHODS |
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Plasmids were linearized with NotI restriction enzyme and transcribed in vitro with T7 RNA polymerase in the presence of the GpppG cap using mMESSAGE mMACHINE kit (Ambion, Austin, TX). Synthetic cRNA was dissolved in water and stored at –70°C before use. Stage V-VI oocytes were obtained by partial ovariectomy of female Xenopus laevis (NASCO, Ft. Atkinson, WI), anesthetized with tricaine methanesulfonate (1.5 g/L, adjusted to pH 7.0). Oocytes were defolliculated by incubation (on a Vari-Mix rocker) in Ca-free modified Barth's solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, adjusted to pH 7.5 with NaOH) containing 2 mg/ml collagenase type IA (Sigma Chemical, St. Louis, MO) for 90 min, and (if necessary) another 90 min in a fresh enzyme solution at 23°C. Oocytes were injected with 0.5 to 1 ng of cRNA and incubated at 19°C in 2x diluted Leibovitz medium (Life Technologies, Grand Island, NY) for 1 to 3 days before measurements were made.
Whole-cell experiments
Whole-cell currents and conductances were measured in intact oocytes using a two-electrode voltage clamp (TEVC) (model CA-1, Dagan, Minneapolis, MN) with 16 command pulses of 30 ms duration between –200 mV and +100 mV, centered around the resting potential. Oocytes expressing ROMK or mutants of ROMK were bathed in permeant acetate buffers to control their internal pH as previously described (14–16). The composition of the bath for the whole-cell experiments was: 50 mM KCl, 50 mM K acetate, 1 M MgCl2, 2 mM CaCl2 5 mM HEPES, and 1 mM SITS (4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid). SITS was used to minimize small endogenous chloride currents; however batches of oocytes exhibiting chloride currents larger than 1 µA were discarded, as were oocytes that did not exhibit at least a 40-mV shift in membrane potential for a 10-fold change in external [K].
Use of permeant buffers to control internal oocyte pH has the disadvantage that control of internal pH requires external pH to be changed as well. Although wild-type ROMK exhibits no significant external pH dependence at the voltages used, some of the mutants did have a measurable external pH dependence. Consequently, all of the two electrode voltage clamp currents were corrected for external pH by first running complete pH cycles with impermeant buffers, in which only outside pH was changed. The same oocytes were then subjected to both internal and external pH changes using permeant acetate buffers. In this way, each oocyte served as its own control, assuming that external and internal pH responses are independent processes.
Control of oocyte pH with permeant buffers required calibration of internal pH as a function of the applied outside pH. A previous calibration for ROMK expressing oocytes resulted in the following linear relationship between external (o) and internal (i) pH: pHi = 0.595 x pHo + 2.4 (16). This calibration was verified in the first section of the Results, by comparing pH titration curves determined with either permeant buffers or excised (inside-out) patches.
Because the mutant channels had somewhat different degrees of inward rectification, pH titration curves were constructed from inward conductances and were normalized to the maximum conductance to compensate for different expression efficiencies between channel types.
All oocytes were preincubated in 100 mM K solutions (50 mM Cl + 50 mM acetate) for 45 min at pH 7.8 to standardize their prior history of K exposure.
Patch-clamp experiments
Cell-attached recordings that were stable for 30 min or longer were used to compare the single-channel properties of the putative glycine hinge mutants to wild-type Kir1.1. In cell-attached mode, internal oocyte pH was controlled with permeant acetate buffers, similar to the two-electrode voltage clamp experiments described above. In addition to acetate, the bath contained 100 mM K and 2 mM Ca and was buffered with 5 mM HEPES to a pH of 7.8. The pipette solution also contained 100 mM K, but no divalents. Osmolarities were adjusted to 205 ± 5 mOsmol/L with NaCl.
Determinations of channel activity were conducted exclusively on inward currents because these give clear, well-defined transitions between open and closed states. The high open probability of Kir1.1 channels made it relatively easy to determine the number of active channels in a given patch. Only patches containing a single channel were used for kinetic analyses.
Excised inside-out patch recordings were used to examine the effect of cytoplasmic-side pH on channel activity in both wild-type and mutant channels. Rundown of channel activity in excised patches was prevented by using FVVP solutions in the bath (17). The composition of FVVP was: 100 mM K, no divalents, 5 mM EDTA, 4 mM NaFluoride, 3 mM Na orthovanadate, and 10 mM NaPyrophosphate, buffered with 5 mM HEPES to the desired pH.
To facilitate comparison between excised patches and intact oocytes, we counted the number of active channels in each excised patch during sequential exposure to different pH's. Because this method unambiguously determines inward rectifier currents, there was no need for leak subtraction (7). Both excised patch activity and inward whole-cell conductance were separately normalized and plotted as functions of internal pH.
Patch-clamp pipettes were pulled from 75 mm borosilicate glass (No. G85165T-3, Warner Instruments, Hamden, CT) using a two-stage process (L/M-3P-A puller) and coated with Sylgard (Dow Corning, Midland, MI). In all patch-clamp experiments, the oocyte vitelline membrane was removed with forceps following a brief exposure to hypertonic (450 mosmol/L) solution.
Pipette resistances ranged from 5 to 10 M
. Currents were recorded with a Dagan 8900 patch-clamp amplifier and stored, unfiltered, on videotape using Instrutech (Port Washington, NY) hardware (VR-10B and ITC-16). Single-channel events were sampled at 5 kHz and analyzed off-line using Bruxton (Seattle, WA) software (Acquire 4.0.10 and Tac/TacFit 4.1.5). The pH titration curves were fitted to a sigmoidal dose-response curve using Prism 4.0c software. All statistical comparisons were conducted using Statview 5.05 software (Cary, NC).
Experiments were performed at room temperature (21 ± 2°C) on Kir1.1b (ROMK2) or mutants of ROMK2 expressed in Xenopus oocytes.
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RESULTS |
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,15,18–22). In these experiments, we used the two electrode voltage clamp to re-measure the pH sensitivity of ROMK in oocytes whose internal pH was modulated with permeant acetate buffers (Fig. 2, solid blue curve). Internal pH was calculated from the calibration given in the Methods section. This curve is in good agreement with previous ROMK pH titrations determined either in cut-open oocytes (22) or with other permeant buffer systems (15).
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To investigate whether acetate buffers were effective at controlling internal pH over a wide range, we compared excised and intact pH titration curves for a mutant (G148A) having a more alkaline pKa than ROMK. Fig. 3 shows inside-out patch recordings of both ROMK and this mutant (G148A-Kir1.1b). Channel rundown was prevented by FVVP in the bath solution (see Methods). Both ROMK and G148A are characterized by high open probability and flickery closures, where the rapid kinetics of the channel reflect interactions between permeant K ions and the selectivity filter (23).
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20 s were required for a 95% change in bath (cytoplasmic side) solution. During this period the exact pH at the internal face of the patch is between the two steady-state pH values given. The single-channel kinetics of wild-type Kir1.1 and the two glycine mutants (G148A-Kir1.1b, G157A-Kir1.1b) are summarized in Table 1. All the kinetic data were obtained on patches containing only a single channel in either the cell attached or excised modes. The kinetics of the two glycine mutants were similar to the kinetics of native ROMK, suggesting that neither the G148A nor the G157A mutation produced major changes in tertiary structure, and did not substantially alter the region around the selectivity filter. However, G148A did have somewhat shorter open times and lower open probabilities than ROMK, whereas G157A had both shorter open and shorter closed times, but no difference in open probabilities (Table 1).
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). Consequently, most of the remaining titrations in this study were determined using the TEVC on intact oocytes, which allowed construction of reliable titration curves, even for mutants having a low expression efficiency.
Role of conserved glycines in pH gating
Conserved glycines in the inner transmembrane helix of both ligand and voltage-gated channels have been implicated as possible hinges in K channel gating (6,8,9,24). Mutating either or both of these glycines did not abolish pH gating, but did alter it significantly, indicating that G148A and G157A are probably both important in the Kir1.1b gating mechanism.
Fig. 4 depicts the intact oocyte pH titration curves for mutations at the two putative glycine hinges of Kir1.1 (rectangles, Fig. 1). Replacing the first glycine by a less flexible alanine residue shifted the pKa for gating by 0.7 pH units from 6.6 ± 0.01 (ROMK) to 7.3 ± 0.01 (G148A-Kir1.1b). Replacing the second conserved glycine (G157) by alanine shifted the apparent pKa by 0.6 pH units to 7.13 ± 0.01 (G157A-Kir1.1b). The combined effect of mutating both glycines to alanine was additive, altering the apparent pKa by 1.2 pH units from 6.6 (ROMK) to 7.8 ± 0.04 for (G148A+G157A). This suggests that the two mutations stabilize the closed state relative to the open state through independent mechanisms.
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The outer helix lysine (K61-Kir1.1b) and pH gating
A lysine (K61-Kir1.1b) near the junction between the outer helix and the slide helix has been implicated as a critical residue for internal pH sensing (14,15,20,22). Mutation of this positively charged lysine to methionine (a residue with a nonpolar side chain) did not abolish the pH sensitivity of the channel but merely shifted it into the acid range (Fig. 5, green line), similar to what is seen with the Kir 2.1 strong inward rectifier (25). In 100 mM K solutions, the K61M-Kir1.1b mutant had a pKa of 5.4 ± 0.05 and a Hill slope of 1.6 ± 0.3, compared to wild-type Kir1.1, which had a pKa of 6.6 ± 0.02 and a Hill slope of between 4 and 5 (Fig. 5, blue line).
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).
If the K61M mutation merely shifts the pKa but leaves the pH-gating mechanism unchanged, then the shift in pKa produced by G157A (relative to ROMK) should be approximately the same as the shift in pKa of G157A-K61M (relative to K61M). In fact, the 
pKa between G157A-K61M and K61M was 1.1 ± 0.04 vs. 0.6 ± 0.02 for the difference between G157A and ROMK (Fig. 5, horizontal dashed arrows). This difference in pKa's implies that the K61M mutation alters a fundamental aspect of the pH gating mechanism.
In the case of the double mutant, G148A-K61M, the K61M mutation clearly dominated over the effect of G148A (Fig. 6). The pKa between G148A-K61M and K61M was 0.08 ± 0.05 vs. 0.72 ± 0.02 for the difference between G148A and ROMK (Fig. 6, horizontal arrows). This effect of K61M on G148A was not unique. Another mutant (I163M), whose pKa was also in the alkaline range, showed a similar interaction with K61M. In this case, the pKa between I163M-K61M and K61M was 0.09 ± 0.05 vs. 0.6 ± 0.03 for the difference between I163M and ROMK (Fig. 6). Hence, the K61 residue appears essential for normal pH gating, even though it is not the exclusive pH sensor for the channel.
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,7). If pH gating of Kir1.1 involves a hinged movement of the inner helix at G148A and/or G157A, mutating the leucine at L160 should disrupt gating not only in wild-type but also in the alkaline shifted mutants, G148A and G157A.
As indicated in Fig. 7, G148A-L160G and G157A-L160G were as insensitive to internal pH as the single L160G mutant (7). Replacement of Leu-160 by the sterically smaller glycine prevented closure of both wild-type and glycine mutants. This suggests that ROMK, G148A, and G157A all utilize the same hydrophobic leucines (L160-Kir1.1b) to close the permeation path at the cytoplasmic end of the inner transmembrane helix.
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DISCUSSION |
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) with the presumed open-state structure of KirBac3.1 (5) implied that gating of Kir channels occurs when hydrophobic residues near the cytoplasmic end of the inner transmembrane helices occlude the permeation path (5–7,10). The mechanism for this closure is thought to involve bending of the inner transmembrane helix at one or more putative glycine hinges, G134 and G143 in KirBac1.1, which are homologous to G148 and G157 in Kir1.1b (1,5,6,8,9,11–13,24,26). However, x-ray diffraction data may or may not depict actual physiological states. Hence, we elected to test the applicability of this glycine-hinge hypothesis to pH gating in the mammalian renal inward rectifier, ROMK (Kir1.1).
Kir1.1 is sensitive to internal pH, with acidification blocking the channel and preventing K efflux across the apical membranes of cortical collecting tubule and thick ascending limb of Henle (14,15,18,21,27). This sensitivity to internal pH was confirmed in this study, using both excised patches and whole-cell currents from ROMK-expressing oocytes.
Previous studies have highlighted the importance of conserved inner-helix glycines in K channel gating (1,5,6,8–10). In Shaker, the single point mutation at G466A (homologous to G148A-Kir1.1b) abolished channel current. However, this may not be entirely due to changes in flexibility at this point in the helix, because G466A also had impaired glycosylation and possibly improper folding in the endoplasmic reticulum (8,24). Less drastic effects of Gly to Ala mutations were reported for the G311A mutation (also homologous to G148A-Kir1.1b) in BK channels (8) and for the G219A mutation in bacterial Na channels (28). The effects of these mutations were similar to the shifts in apparent pKa produced by the Kir1.1b glycine mutations, reinforcing the notion that inner-helix glycines are important, but not absolutely essential for Kir gating.
In our studies on Kir1.1, mutation of both conserved inner helix glycines to alanines did not abolish pH gating but alkaline-shifted the apparent pKa in an additive fashion. Namely, the combined effect of G148A and G157A (= 1.2 pH units) was close to the sum of the individual mutations: G148A (= 0.7 pH units) and G157A (= 0.6 pH units). This suggested that both glycines contribute independently to the gating process.
The failure of either G148A or G157A to completely abolish gating in Kir1.1b clearly shows that gating does not absolutely require the presence of glycine hinges at either of these positions. One possibility is that the alanines used to replace the conserved glycines allow sufficient angular movement of the inner helix so that the mutants still gate, but at a higher pKa. It is also possible that a hinge motion is not required for gating. Electron paramagnetic resonance studies of KcsA gating transitions suggest that a twisting and scissor-like movement of the inner transmembrane helices may be predominant steps for opening this channel. In fact, the region near residues 107, 108 on KcsA (corresponding to the G157 locus in Kir1.1b) was implicated as a pivot point for helix movement in KcsA (29,30). In this interpretation, the effect of the alanine for glycine substitution might be due to the larger alanine side chain introducing a steric hindrance to channel opening.
K61-Kir1.1b and pH gating
The finding of pH gating in the K61M mutant (Fig. 5) makes it unlikely that the K61-Kir1.1b (K80-Kir1.1a) residue functions as the exclusive pH sensor for the channel, as previously envisioned (31). However, K61 might still play a sensor role if its free solution pKa of 10.5 were brought closer to 7 by interaction with either positive residues (19) or the membrane lipid environment, since K61 is now known to reside within the membrane, rather than in the cytoplasm (6).
Our data indicate that G148A and G157A react differently to the K61M mutation. The double mutation of G157A and K61M produced a titration curve midway between the individual curves for G157A and K61M and close to the value of the wild-type channel (Fig. 5). This suggests that G157A may be acting independently of the K61M mutation, with both mutations contributing oppositely (and about the same magnitude) to the final titration curve for G157A-K61M. This is in contrast to the double mutant G148A-K61M, which resulted in a titration curve that was indistinguishable from the K61M titration alone (5.5 ± 0.02 vs. 5.4 ± 0.05), and suggested a dominance of the K61M mutation over the G148A mutation (Fig. 6).
We also examined the effect of K61M on the I163M mutant, which was previously shown to gate in a more alkaline range than wild-type ROMK (7,32). The effect of the K61M mutation on I163M was similar to its effect on G148A, where the pKa of K61M-I163M was indistinguishable from that of K61M-G148A (Fig. 6).
These data imply that K61 is not absolutely essential for pH gating but could act as one of several pH sensors, in this case operating in the range above pH 6; whereas other pH sensor(s) might function at an internal pH below 6. Alternatively, K61 might be acting as an intermediary link between a cytoplasmic pH sensor and the putative physical gate at the bundle crossing of the inner helices (33).
Both G148A and G157A still rely on the putative gate at L160G
Regardless of how G148A and G157A alter pH gating, both of these glycine mutants still utilized leucine residues at L160-Kir1.1b to physically occlude the permeation path. Evidence for this comes from the finding that the double mutants, L160G-G148A and L160G-G157A, remained open at low internal pH, similar to the single point mutant, L160G-Kir1.1b.
Furthermore, the acid-shifted pH gating of the K61M mutant also depended on these same hydrophobic leucines at L160, since the double mutant L160G-K61M failed to close, even at extremely low pH (Fig. 5). Previous work had indicated that replacing the leucines at L160-Kir1.1b with residues having small or highly polar side chains allowed passage of hydrated K ions at low pH, when the gate would normally be closed (7). Hence, this study confirms a physiological gate at L160-Kir1.1b that is functionally separate from any bending of the inner helix, as well as from the pH sensor itself.
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SUMMARY |
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,6) and electrophysiological (7) data, suggesting that pH gating occurs via steric occlusion of the permeation path by four leucine side chains at the helix bundle crossing, which is facilitated by conserved glycines on the inner transmembrane helix. However, pH gating in Kir1.1 does not absolutely require glycine hinges in this region. |
ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health grants DK46950 (H. Sackin) and DK27847 (L. G. Palmer).
Submitted on October 25, 2005; accepted for publication February 2, 2006.
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REFERENCES |
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