Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR
EVIDENCE FOR DISRUPTION OF A SALT BRIDGE*

Joseph F. Cotten and Michael J. WelshDagger

From the Howard Hughes Medical Institute and Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

    ABSTRACT
Top
Abstract
Introduction
References

Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wild-type CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction.

    INTRODUCTION
Top
Abstract
Introduction
References

The cystic fibrosis transmembrane conductance regulator (CFTR)1 contains an anion-selective pore (1-6). Earlier work has shown that amino acid residues in the two membrane-spanning domains, MSD1 and MSD2, determine the properties of this pore and harbor a number of disease-associated mutations (7-12). Despite the importance of this region to CFTR function, an understanding of the pore and MSD structure is limited.

Studies of the effect of cystic fibrosis (CF)-associated mutations have been of value in identifying structurally and functionally important regions of CFTR. At least four CF-associated mutations have been identified at position 347 in M6: R347C, R347H, R347L, and R347P, suggesting that Arg-347 is important for CFTR structure and function (13-15).2 Early studies by Sheppard et al. (7) showed that mutation of Arg-347 to proline significantly decreased single-channel conductance with little effect on CFTR trafficking to the plasma membrane. Other work by Tabcharani et al. (8, 16) and Linsdell and Hanrahan (17) emphasized the importance of Arg-347 for anomalous mole-fraction behavior, iodide permeability, and voltage-dependent block by DIDS, in addition to single-channel conductance. Interestingly, mutation of Arg-347 to a histidine (R347H) produced a channel that displayed pH-dependent conductance and anomalous mole-fraction behavior (8). These studies suggested that Arg-347 may line the pore and that a positive charge at position 347 is sufficient for wild-type conductance. Because mutation of Arg-347 eliminated anomalous mole-fraction behavior, Arg-347 itself was proposed to be an anion binding site in the CFTR pore (8), and the presumed positive charge introduced upon protonation of His-347 was thought to facilitate interactions with permeating anions. An alternative interpretation is that Arg-347 may be important for maintenance of pore architecture without contributing directly to the permeation pathway. For example, mutation of this site could lead to a change in MSD conformation and loss of an anion-binding site(s) elsewhere; protonation of His-347 might then rescue the conformation of the R347H mutant.

As discussed by Perutz (18), charged residues within proteins reside in locations where they are either solvated or can interact with and be neutralized by oppositely charged residues. These electrostatic interactions mediate an important stabilizing effect, providing increased thermostability and resistance to denaturation. Based on these considerations, it is possible that Arg-347 may line the pore where it can interact with either water or permeant anions. However, it is also possible that Arg-347 may mediate a structural role in the MSDs; there are a number of negatively charged residues with which Arg-347 might interact. Both possibilities are consistent with the present data.

To better understand the role of Arg-347 in CFTR structure and function, we examined the effect of mutating Arg-347 to cysteine, aspartic acid, glutamic acid, lysine, and leucine on CFTR conductance. We examined the cytosolic pH (pHc)-dependent behavior of CFTR-R347H and that of the other residue 347 mutants both with (R347C, R347D, R347E, and R347K) and without (R347L) a pHc-titratable residue. The conductance of CFTR-R347H is pHc-dependent. Because the site of protonation may exist in one of two states, either protonated or deprotonated, we tested the hypothesis that CFTR-R347H may display two pHc-dependent conductance states, which it did. If the protonatable site lines the pore, then the two ionization states of the protonatable residue might yield two distinct conductance states. Alternatively, if the protonatable site influences structure, then the two conductance states might represent two distinct conformational states of CFTR. Moreover, like CFTR-R347H, all the other residue 347 mutants, except R347K, displayed two pHc-dependent conductance states over a similar pHc range. The residue at position 347 did not influence current flow through either conductance state. These data suggested that residue 347 probably does not line the pore but likely stabilizes CFTR structure. To pursue this, we studied the effect of mutating residues elsewhere in the MSDs that might interact with Arg-347.

    EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and Transfection-- All mutants were constructed in the pTM1-CFTR4 plasmid by the method of Kunkel (19). The mutagenesis was confirmed by restriction digestion of silently introduced restriction sites and by sequencing around the introduced mutation site. In vitro transcription and translation of each mutant was performed to confirm expression of full-length protein. Wild-type and mutant channels were expressed transiently in HeLa cells using the vaccinia virus/T7 bacteriophage hybrid expression system as described previously (20). Cells were routinely studied 12 to 24 h after infection-transfection.

Patch-Clamp Technique-- Methods used for excised, inside-out patch clamp recordings were as described previously (21-23). Voltages were referenced to the extracellular side of the membrane. All studies were done at room temperature (22-24 °C) to facilitate kinetic analysis. The membrane potential was clamped at -120 mV unless otherwise indicated.

CFTR was activated by excising patches into a bath solution (pHc 7.3; Tricine or TES) containing 1 mM ATP and 75 nM catalytic subunit of cAMP-dependent protein kinase (Promega Corp., Madison, WI). During the pHc studies, cAMP-dependent protein kinase was removed, and bath (cytosolic side) ATP concentration was adjusted (usually to less than 0.05 mM ATP) to resolve single-channel bursting activity within multichannel macropatches. For experiments with excised, inside-out patches, the pipette (extracellular) solution contained (in mM): 140 N-methyl-D-glucamine (NMDG), 140 aspartic acid, 10 Bis-Tris, 5 CaCl2, 2 MgSO4, pHo 6. The bath (intracellular) solution contained (in mM): 140 NMDG, 10 Bis-Tris, 3 MgCl2, 4 CsOH/1 EGTA, pHc 6.5 (unless indicated) with HCl ([Ca2+]free < 10-8). We selected Bis-Tris as a buffer to avoid the blocking effect that Good's type buffers such as MOPS have on CFTR (24). pH was adjusted at room temperature (22-24 °C) using a Model 10 Accumet meter fitted with a gel-filled combination electrode calibrated with pH 6.0 or 7.0 standardized buffer solutions, where appropriate (all pH analytical equipment was from Fisher Scientific, Pittsburgh, PA).

Data Analysis-- Single-channel current amplitudes were determined from the peaks in all-points histograms. Single-channel conductances were derived from the slope of the single-channel linear I-V relationship. Single-channel data were filtered at 1000 Hz using an 8-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA), digitized at 5000 Hz, and digitally filtered at 500 Hz. The resolution of the A/D converter was 0.0012 pA. Events lists were generated using a half-height transition protocol; transitions less than 1 ms were excluded. For events list generation, a prompt was positioned by eye at each current level representing the closed state, the little conductance state (OL), and the big conductance state (OB). The lifetimes of individual sojourns in OL or OB were collected, binned (10 bins per decade), and fitted with a one-component exponential using the maximum likelihood method. Wild-type CFTR enters a short-lived intraburst closed state; for simplicity we omitted this state in our analysis of residue 347 mutants since it is rare in wild-type CFTR in the absence of MOPS or other blocking buffers (approximately once every 213 ms within bursts of duration equalling 649 ms; n = 2 at pHc 6.0, -120 mV, 22-24 °C) on the time-scale of transitions between the OL and OB state (24). Single-channel current variance analysis was done as described in the legend for Fig. 3 and in Ref. 25. Data acquisition and analysis were done using the pClamp software package (Axon Instruments Inc., Foster City, CA) and Excel 5.0 (Microsoft Corp., Redmond, WA).

    RESULTS

pHc-dependent Conductance of Residue 347 Mutants-- To determine whether R347H exhibits two discrete conductance states, we studied single channels in excised, inside-out patches. Fig. 1 shows a representative single-channel trace from R347H displaying two pHc-dependent conductance states, OL and OB. Unexpectedly, all the other variants at residue 347, except R347K, showed two pHc-dependent conductance states over a similar pH range. Even R347L, which does not have a titratable side chain, displayed this behavior (Fig. 1). The conservative substitution of Arg-347 by Lys (R347K) showed only a single conductance state and no pHc dependence. Similar results were observed when NMDG-Cl was replaced with NaCl or when Bis-Tris was replaced with MES (n = 2 each, not shown), suggesting that this behavior is not due to block by a pHc-dependent change in one of the pH-titratable bath reagents, NMDG or Bis-Tris. The pH of the pipette solution did influence the results (not shown). Also, there was no state-dependent bias in opening or closing. Channels opened into or closed from the OL or OB states as predicted based solely upon pHc.


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Fig. 1.   Single-channel current tracings from excised, inside-out membrane patches containing the wild-type or indicated residue 347 mutant channels. All current tracings were collected with membrane voltage clamped at -120 mV at the indicated intracellular pHc; the pipette pHo was 6.0 throughout. All-points histograms were derived from 7-40 s of data using a binwidth of 9.8 or 19.5 fA and are shown to the right of current tracings. C refers to closed state.

The all-points histograms (Fig. 1) illustrate qualitatively that as pHc increased, each mutant except R347K spent an increased fraction of time in OL and a correspondingly decreased fraction of time in OB. Points in the histogram from the zero current state (C), which are largely a function of ATP concentration (26-28), are included only for purposes of clarity. Wild-type CFTR and R347K possess only one predominant conductance state over the pHc range 5.5-7.3 (Fig. 1, data not shown, and Ref. 8). This result suggests that a large positively charged residue at position 347 (Arg or Lys) in CFTR stabilizes the OB state relative to the OL state. Visual inspection suggested that the lifetimes of OL and OB states were also influenced by the nature of the residue at position 347: R347E and R347H tended to have longer dwell times in the OL and OB states, whereas R347L, R347C, and R347D tended to display shorter dwell times. In general, the smaller the residue, the more rapid the kinetics. Perhaps steric hindrance imposed by the residue at position 347 might explain the size dependence of the kinetics. For R347D, the lifetime in the OB state was so short that a discrete OB was not apparent on the all-points histogram; instead, as pHc decreased, a shoulder developed on the OL state distribution in the all-points histogram (Fig. 1). Since R347L displayed two pHc-dependent conductance states and since leucine is aliphatic and non-ionizable, the pHc dependence of residue 347 mutants cannot be attributed simply to protonation of residue 347.

Single-channel Conductance of Residue 347 Mutants-- To determine whether the residue at position 347 affects single-channel conductance and not merely the conductance state of the channel, we examined the I-V relationship and slope conductance of the mutants with slower pHc-dependent kinetics, R347H and R347E, as well as R347K. Filtering obscured the true current amplitudes of the other pHc-dependent mutants with more rapid kinetics. Fig. 2 shows that the I-V relationships and slope conductances for the OL and OB states were not significantly affected by the nature of the residue at position 347. The single-channel conductance at pHc 6.0 of wild-type CFTR, R347K, and the OB state of R347E and R347H were all very similar (in pS): 7.7 ± 0.4, 8.3 ± 0.6, 7.4 ± 0.4, and 6.9 ± 0.2, respectively (n = 3 for each). The single-channel conductance of the OL states of R347E and R347H at pHc 6.0 were also very similar (in pS): 1.5 ± 0.1 and 1.6 ± 0.1, respectively (n = 3 and 4 for each). These data suggest that the amino acid at residue 347 does not affect single-channel current amplitude, rather the predominant effect is on the lifetime of the OL and OB conductance states. The lack of effect of the specific residue at position 347 on the rate of Cl- flow through the pore in either the OL or OB conductance state suggests that residue 347 does not interact directly with permeating anions.


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Fig. 2.   Single-channel I-V relationships for R347E (OL and OB states), R347H (OL and OB states), R347K, R347D/D924R, and wild-type CFTR at pHc 6.0. n = 2-4 at each data point.

There are at least two possible explanations for the pHc-dependent behavior of residue 347 mutants. One explanation is that mutations at residue 347 reveal the effect of a protonatable residue in the permeation path that directly interacts with anions. In this case, the lifetime of the OB and OL state represent the lifetime of the protonated and deprotonated ionization states of this unknown site, respectively. A second explanation is that when Arg-347 is mutated, the membrane-spanning domains that form the pore fluctuate between two conformational states, OL and OB; protonation or deprotonation of some unknown site favors the OB or OL conformational states, respectively. The first explanation seems unlikely because the reciprocal lifetime of the OL state which represents the "on" rate for the proton is very slow (e.g. it is 6 × 107 M-1 s-1 for R347E). This rate is ~200-fold slower than proton transfer onto an imidazole in free solution (29, 30) and ~10-fold slower than transfer onto an imidazole in the pore of ROMK1 (25). The slow rate, however, might be explained in part by residence of the protonatable site in a sterically and electrostatically shielded position. In addition, the site of protonation revealed by a residue 347 mutation would have to perfectly replace the lost anion-binding site incurred through the mutagenesis; this seems exceedingly fortuitous. This consideration convinced us to favor a model involving a conformational change in CFTR.

Dwell-time Analysis of OL and OB States-- We performed a dwell-time analysis of the lifetimes of the OL and OB states of R347E and R347H to enable more quantitative comparisons between them and to better understand their pHc dependence. Both OL and OB dwell-time histograms were best fit by a single exponential function at all pHc values examined. This suggests that the distribution between OL and OB state does not change markedly throughout the gating cycle; this can also be observed visually in Fig. 1. Fig. 3A shows that the reciprocal lifetime of the OL state increased relatively linearly with increasing proton concentration. This indicates that the rate of entry into the OB state increased with increasing proton concentration. This linear, first order dependence on proton concentration suggests that protonation of only a single site limits the rate of movement from the OL to the OB state. The rate of exit from the OB state decreased non-linearly with increasing proton concentrations. The kinetics and the equilibria between OL and OB were very similar for both mutants (Fig. 3A). The observable pK (0 mV) for the equilibrium between OL and OB of R347E and R347H were 6.4 and 6.3, respectively. The faster kinetics of R347D, R347C, and R347L made dwell-time analysis for these mutants less reliable. Therefore, to obtain an estimate of the approximate pK of these mutants, we examined changes in the variance of the open-state current with changes in pHc (25). The current variance should go through a maximum when the pHc equals the observable pK. Each mutant displayed an increase in current variance with increasing proton concentration (i.e. decreasing pH). Fig. 3B shows that R347C, R347D, and R347L did not reach a peak variance over the range of pHc studied, suggesting that their apparent pK is less than 5.0-5.5. Visual inspection of the tracings suggested that the mutants were largely in their OL state over the range of pHc employed such that we did not miss a peak in the variance. In this analysis, we assumed that the mutants would display minimal pH-independent movement between OL and OB states at very acidic pHc so that the variance should pass through a maximum as pHc decreases. As a control for the variance analysis, we examined the R347E mutant on which we had also done dwell-time analysis (Fig. 3A); as expected, Fig. 3B shows that the variance of R347E goes through a maximum between pHc 6.5 and 5.5. 


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Fig. 3.   pHc dependence of residue 347 mutants. A, dwell-time analysis in the OL and OB conductance states versus pHc for R347E and R347H. n equals 3-7 for each. B, open-channel current variance of the R347C, R347D, R347L, and R347E mutants versus pHc. Open-channel current variance was calculated by subtraction of current variance in the closed state (sigma c2) from current variance in the open state (sigma o2). Each data point was derived from 2-4 excised, inside-out membrane patches; in general, 10 measurements (sigma o2 - sigma c2) were averaged per patch and were obtained from individual openings and adjacent closed periods. Error bars are smaller than the symbols.

Voltage Dependence of OL and OB-- To learn more about the nature of the two conductance states of residue 347 mutants, we examined the voltage dependence of the OL and OB lifetimes. Fig. 4A shows qualitatively that both conductance states of R347E were voltage-dependent. Fig. 4B shows quantitatively that at pHc 6.0 the OL and OB states for R347E and R347H were both influenced by the transmembrane voltage. The voltage dependence may derive from alterations in the distribution of protons near the protonatable site or from charged regions of CFTR moving through the voltage field. We assumed the Boltzman distribution to quantify the effect of voltage on the equilibrium between OL and OB states such that
<UP>p</UP>K(<UP>mV</UP>)=<UP>p</UP>K(0 <UP>mV</UP>)+&thgr;zFE/2.303RT (Eq. 1)
in which theta  = electrical distance from the cytosolic surface, z = valence, F = Faraday's constant, E = transmembrane potential, r = gas constant, T = temperature, pK = the negative log of the equilibrium constant between OL and OB conformations. The degree of voltage dependence was similar for both mutants despite the charge differences at residue 347 and yielded a theta z of 0.25 and 0.21 for R347E and R347H, respectively. The voltage dependence was asymmetrically disposed between the rate of entry into the OB state and the rate of exit from OB (Fig. 4B). To quantify these differences we used the following:
&tgr;<SUB><UP>L</UP></SUB><SUP><UP>−</UP>1</SUP>(<UP>mV</UP>)=&tgr;<SUB><UP>L</UP></SUB><SUP><UP>−</UP>1</SUP>(0 <UP>mV</UP>) · <UP>exp</UP>[(1−&dgr;)&thgr;zFE/RT] (Eq. 2)
&tgr;<SUB><UP>B</UP></SUB><SUP><UP>−</UP>1</SUP>(<UP>mV</UP>)=&tgr;<SUB><UP>B</UP></SUB><SUP><UP>−</UP>1</SUP>(0 <UP>mV</UP>) · <UP>exp</UP>[(<UP>−</UP>&dgr;)&thgr;zFE/RT] (Eq. 3)
in which delta  = symmetry factor which partitions voltage dependence (31, 32). The rate of exit from the OB state (tau B-1) was more voltage-dependent than the rate of exit from the OL state (tau L-1) for both mutants (delta  = 0.8 versus- delta  = 0.2 for R347E and delta  = 0.7 versus- delta  = 0.3 for R347H). This observation may be explained by the protonatable site moving through the voltage field; protonation and deprotonation of this site before and after the conformational change will affect the net charge migrating through the applied voltage. Since voltage dependence arises from charge movement through a voltage field and since R347E and R347H displayed similar voltage dependences and carry different charges at position 347, residue 347 is not likely moving through a transmembrane potential during interchange between OL and OB states.


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Fig. 4.   Voltage dependence of OL and OB states. A, current records from excised, inside-out membrane patch containing R347E channel. Traces were collected at pHc 6.0 and pHo 6.5 at the indicated transmembrane voltage. B, dwell times (pHc 6.0) in the OL state (closed symbols) or OB state (open symbols) versus voltage for R347E (left, circles) and R347H (right, squares). Error bars are smaller than the symbols. n equals 4-6 for each.

The Phenotype of R347D Is Suppressed by the D924R Mutation-- The data suggest that Arg-347 and Lys-347 may stabilize the structure of the pore; in their absence, the channel "flickers" between two conductance states. Arginine and lysine residues through electrostatic interactions with anionic residues are important for the structure of membrane-spanning domains in other proteins such as the Lac permease and the inward-rectified K+ channel, IRK1 (33-35). As discussed by Perutz (18), salt bridges within proteins are an important structural feature that confers thermostability and resistance to denaturation. We hypothesized that Arg-347 may mediate a stabilizing influence by contributing to a salt bridge within the MSDs. There are multiple glutamates and aspartates within transmembrane (M) regions with which Arg-347 or Lys-347 might interact: Glu-92 (M1), Glu-873 (M7), Asp-924 (M8), Asp-993 (M9), and Glu-1104 (M11). To identify the Arg-347 interaction partner, we replaced Arg-347 with an anionic residue (R347E or R347D) and introduced an arginine residue in the place of candidate partners in a salt bridge. We studied the conductance properties of the following double mutants: R347D/D924R, R347D/D993R, and R347E/E1104R. The R347D/D993R and R347E/E1104R mutants each had two conductance states with pHc-dependent behavior (Fig. 5). For R347D/D993R the increased entry into the OB state was apparent as a shoulder on the amplitude histogram at pHc 5.5. Accordingly, for R347D/D993R and R347E/E1104R the current variance in the open state increased with decreasing pHc (Fig. 5B). Qualitatively, the lifetimes of the OL and OB conductance states in the R347D/D993R and R347E/E1104R were similar to that of the R347D and R347E mutants, respectively. The amplitude of the OL state was larger for both of these double mutants as compared with the single mutants (Figs. 1B and 5B). We also observed an infrequent, additional small conductance state in the R347E/E1104 mutant (see amplitude histogram in Fig. 5A); this is likely due to the E1104R mutation itself.


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Fig. 5.   A, single-channel current tracings from excised, inside-out membrane patches containing R347E/E1104R, R347D/D924R, and R347D/D993R. Membrane voltage was -120 mV, and pHc is indicated; pipette pHo was 6.5 throughout. All-points histograms were derived from 6-39 s of data using a binwidth of 9.8 or 19.5 fA and are shown on the right. B, current variance of R347E/E1104R, R347D/D924R, and R347D/D993R at the indicated pHc was collected as in Fig. 3. Each data point was derived from 2-3 patches. Error bars are smaller than the symbols.

In contrast to the other double mutants, the R347D/D924R mutant did not display the pHc-dependent flicker found in the R347D single mutant (Fig. 5, A and B), and there was no effect of pH on open-channel variance (Fig. 5B). The single-channel conductance was similar to that of wild-type CFTR (6.1 ± 0.1 pS; n = 3; Fig. 2). These data suggest that the D924R mutation compensates for or rescues the phenotype of the R347D mutation. This result predicts that the D924R mutation alone (with Arg at position 347) would generate an unstable channel with at least two open conductance states. Fig. 6 shows that the D924R single mutant displayed multiple (~3) conductance states that appeared to be pHc-independent.


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Fig. 6.   Single-channel currents from the D924R variant. Currents were obtained at indicated pHc with membrane voltage maintained at -120 mV. Similar results were obtained in three patches.


    DISCUSSION

Function of Arg-347 in CFTR-- Previous work from our and Hanrahan's laboratories (7, 8, 17) has led to the speculation that Arg-347 may be an anion-binding site in the CFTR pore. However in contrast to earlier interpretations, our current data show that residue 347 does not influence permeation properties via a direct interaction with permeating anions. Instead they suggest that Arg-347 may be more important for maintenance of pore architecture.

We found that mutation of residue 347 to glutamate, aspartate, cysteine, histidine, or leucine all produced channels with two distinct conductance states, OL and OB. pHc, and voltage influenced the movement between these two states over a similar range for all mutants. Additionally, the single-channel slope conductances of R347H and R347E were the same in both OB and OL states. The OB state for each had the same conductance as wild-type CFTR. The average calculated pKa for a glutamate and a histidine within a protein are 4.0 and 6.9 (36), respectively. If His-347 or Glu-347 line the permeation pathway, at pHc 6.0 (bath solution) or pHo 6.5 (pipette solution) His-347 is predicted to be protonated and to have at least a partial positive charge, and Glu-347 should be fully deprotonated and have a negative charge. Since the charge or the structure of the residue at position 347 failed to affect single-channel conductance, it seems unlikely to be either an anion-binding site or to be positioned such that it interacts sterically or electrostatically with permeating anions. Thus, mutation of Arg-347 may decrease single-channel conductance and anomalous mole-fraction behavior by disrupting pore architecture and the function of some other anion-binding site(s).

The OB and OL Conductance States-- All the mutants except R347K showed two pH-dependent conductance states. The equilibria between OB and OL states were similar despite the nature of the mutation at residue 347 with values of pK ranging from ~5-7.3 Because the values of pK were roughly similar, the data suggest that the structure, titratability, and pK of residue 347 do not markedly influence the distribution between OL and OB states. The linear dependence on proton concentration for OB entry suggests that a single protonatable site largely determines entry into this state.

The OB and OL states may represent two protonation states of the CFTR molecule or two distinct conformational states that are influenced by protonation. For the reasons discussed above, we favor the latter possibility, that alternating residence in two discrete conformations is responsible for the two conductance states. How might this occur? We hypothesize that Arg-347 forms a salt bridge with another negatively charged residue in CFTR. Mutation of Arg-347 would leave a negatively charged residue unpaired within the membrane. Charged molecules within a low dielectric constant are unstable (37). Therefore, the protein might reorient to solvate the charge, i.e. enter the OL state. Subsequent protonation of that site would neutralize its charge and allow the protein to reorient back to its native conformation, i.e. return to the OB state.

This model suggests that the residue(s) with which Arg-347 interacts may be the protonatable site. Several observations are consistent with this hypothesis. First, we found that a positively charged lysine, which can support a salt bridge, was able to supplant arginine. However, the other residues tested were not able to substitute for arginine. Perhaps histidine was not able to replace arginine because its side chain was not long enough or it may have an anomalously acidic pKa within the low dielectric constant of the membrane.

Second, and more importantly, we found that a second-site complementary mutation at position 924 (D924R) largely eliminated the pHc-dependent flickering phenotype of the R347D mutation and restored current amplitude to near wild-type values. We also recognize the possibility that Arg-347 may interact with additional yet untested residues, e.g. Glu-873 in M7.

Third, a salt bridge between Arg-347 and Asp-924 should be disrupted by mutation of Asp-924. As predicted, we found that the D924R mutant displayed erratic flickery, pHc-independent behavior. Presumably, this mutation also generates an unstable channel. The pHc independence of D924R is also consistent with the hypothesis that Asp-924 is the site of protonation in the residue 347 mutants. Interestingly, mutation of a glutamate in the putative salt bridge in the P-loop of the IRK1 channel leads to a single-channel flickering phenotype reminiscent of residue 347 mutations (35).

Tertiary Structure of the Membrane-spanning Domains-- At the simplest level, the data suggest that both MSDs functionally interact in a manner that influences permeation. The studies of R347D/D924R are consistent with a salt bridge between Arg-347 and Asp-924 and thus an interaction between M6 and M8. This further suggests that mutations in MSD2 may alter the phenotype of mutations in MSD1. Consistent with this, mutation of D993R and E1104R in MSD2 increased the relative amplitude of the OL conductance state in the context of Arg-347 mutations.

The Arg-347 residue is targeted by several CF-associated mutations, R347C, R347H, R347L, and R347P (13-15).2 Our data suggest that CF-associated as well as other mutations at residue 347 affect CFTR similarly. They disrupt pore architecture by disrupting an interaction between Arg-347 and another residue(s), one possibly being Asp-924 in M8. These data highlight the importance of residue 347 for CFTR function and may explain in part why this residue is targeted by multiple CF-associated mutations.

    ACKNOWLEDGEMENTS

We thank Pary Weber, Lisa DeBerg, and Terri Grunst for excellent technical assistance. We thank our laboratory colleagues as well as Drs. David Gadsby, Toshinori Hoshi, and David Sheppard for helpful discussions.

    FOOTNOTES

* This work was supported by Grants HL29851 and HL42385 from the NHLBI, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 500 EMRB, Iowa City, Iowa 52242. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail: mjwelsh{at}blue.weeg.uiowa.edu.

2 C. Ferec, personal communication.

3 The shift toward a lower pK for mutants with more rapid kinetics (Fig. 1 and 3B) may be a filtering artifact; from analysis of the R347E mutant and the R347H mutant, we know that the OB state tends to be shorter then the OL state at the applied transmembrane voltage and is therefore more vulnerable to filtering. We presume that this relationship is preserved throughout all the mutants.

    ABBREVIATIONS

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; M, transmembrane domain; MSD, membrane-spanning domain; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; MOPS, 3-(N-morpholino)propanesulfonic acid; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; Tricine, N-tris(hydroxymethyl)methylglycine; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; NMDG, N-methyl-D-glucamine; I-V, current-voltage relationship; pHc, cytosolic, bath pH; pHo, extracellular, pipette pH; OL, little conductance state; OB, big conductance state; tau L, lifetime of little conductance state; tau B, lifetime of big conductance state.

    REFERENCES
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Abstract
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