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CFTR: A Cysteine at Position 338 in TM6 Senses a Positive Electrostatic Potential in the Pore

Xuehong Liu ^*, Zhi-Ren Zhang , Matthew D. Fuller , Joshua Billingsley ^*, Nael A. McCarty  and David C. Dawson ^*

^* Department of Physiology/Pharmacology, Oregon Health & Science University, Portland, Oregon; and School of Biology, Georgia Institute of Technology, Atlanta, Georgia

Correspondence: Address reprint requests to David C. Dawson, Dept. of Physiology/Pharmacology, L334, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239. Tel.: 503-494-2803; Fax: 503-494-4352; E-mail: dawsonda{at}ohsu.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in g_Cl and I-V shape that were specific to the substituted cysteine. The apparent pK_a of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pK_a was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET⁺ and MTSES^– were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pK_a of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
The anion selective pore of the CFTR chloride channel has been probed by means of site-directed mutagenesis and covalent modification of engineered cysteines (Akabas, 1998; Cheung and Akabas, 1996, 1997; Smith et al., 2001). Based on the permeation of halides and tighter-binding, polyatomic anions (often referred to as pseudohalides; Douglas et al., 1994) we proposed that the CFTR channel could be envisioned as an hourglass-shaped pore comprising a narrow region, where anions bind, coupled to the external and internal solutions by two wider vestibules (Smith et al., 1999). Modification of engineered cysteines by pH and thiol-directed reagents were consistent with the hypothesis that R334 in transmembrane segment 6 (TM6) lies in the outer vestibule of the pore, where it establishes a positive electrostatic potential that increases the local concentration of Cl^–, thereby enhancing pore conductance (Smith et al., 2001). Here we present evidence that T338, which lies one (predicted) helical turn cytoplasmic to R334 (Fig. 1), also resides within the pore. Furthermore, we propose that the titration of a cysteine residue at 338, as well as the rate of modification of this cysteine by charged thiol-directed reagents, provides a crude measure of a positive electrostatic potential in this region of the pore that may be attributable to R334 and perhaps other residues in the outer vestibule.

View larger version (30K): [in this window] [in a new window]   FIGURE 1  Predicted topology for CFTR with TM6 enlarged to show the individual residues. Highlighted with shaded background are T338, the four endogenous basic amino acid residues (R334, R335, R347, and R352, solid circles), and an endogenous cysteine (C343, shaded circle). T338 is approximately one helical turn cytoplasmic to R334.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
Mutagenesis and in vitro transcription
The methods used for mutagenesis and in vitro transcription were the same as those reported previously (Smith et al., 2001; Liu et al., 2001). CFTR mutants were generated using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The sequences in the region of the mutation and in the whole PCR-generated region were confirmed by direct DNA sequencing.

The CFTR cRNAs for Xenopus oocyte injection were synthesized by using the in vitro transcription kit, mMessage mMachine (Ambion, Austin, TX). The transcription products were purified and the quality and quantity of the transcripts were assessed on an agarose gel.

Oocyte preparation
Protocols for preparing oocytes were similar to those previously described (Smit et al., 1993; Wilkinson et al., 1996). Oocytes were surgically removed from anesthetized South African clawed frogs, Xenopus laevis, and manually defolliculated after incubation in a collagenase-containing solution for 2–2.5 h. The oocytes were kept in an 18°C humidifier overnight in a modified Barth's Solution containing 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂ · 4H₂O, 0.41 mM CaCl₂ · 2H₂O, 2.4 mM NaHCO₃, 10 mM HEPES-HemiNa, and 250 mg/L Amikicin. Oocytes were then injected with 50 nL cRNA at 0.2 ng/nL with or without the cRNA encoding human ß₂-adrenergic receptor. Typically, oocytes were used within five days after injection.

Electrophysiological recordings
Oxidation states of the cysteine at 338
Preliminary experiments indicated that the oxidation state of the cysteine substituted at position 338 varied spontaneously, as evidenced by a variable effect of reducing agents (2-ME or DTT) to increase conductance in oocytes expressing this construct (Liu et al., 2002). Accordingly, care was taken to promote the reduced state of the cysteine by pretreating oocytes with 1 mM 2-ME or DTT before, or during, recordings. This concentration of reducing agents produced maximal effects in preliminary experiments. Although the observed reactivity of this construct toward MTS reagents and NEM (see Results) suggested that this strategy was generally successful, we cannot eliminate the possibility that in a small fraction of the channels, the cysteine thiolate was oxidized to a state that was not susceptible to reducing agents, e.g., a sulfinic (SO₂H) or sulfonic (SO₃H) acid (Giles et al., 2003; Jacob et al., 2003). We refer to these nonreducible states as terminal oxidation states.

Whole-cell recordings
The methods were similar to those described by Mansoura et al. (1998). Briefly, individual oocytes were placed in the recording chamber and continuously perfused with Frog Ringer solution containing 98 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES-HemiNa, pH 7.4. For those experiments in which the bath pH was modified, HEPES was replaced with MES (for pH 6.7 or less), TAPS (for pH 7.7–9.1), or CAPS (for pH 9.7–11). The different buffers produced no discernible changes in the electrical behavior of wt CFTR (Smith et al., 2001). The volume of the perfusion chamber used in the current study was 100 µL and the flow rate to the chamber was 67 µL/s (4 mL/min). The room temperature was between 19 and 24°C. A two-electrode voltage-clamp system (TEVC-200, Dagan, Minneapolis, MN) was used for data acquisition. Oocytes were normally maintained in experimental chambers under open circuit conditions. At the time of interest, the membrane potential was ramped from –120 mV to +60 mV in a period of 1.8 s to construct the whole cell I-V plots. The I-V data recorded from each oocyte were analyzed using a program developed in our laboratory and the conductance was calculated from the slope of the I-V plot at the reversal potential (g_Cl at V_m = E_rev) using a voltage range from V_m = E_rev –10 mV and V_m = E_rev +10 mV. Data are reported as mean ± SE.

CFTR channels were activated using a cocktail containing 1 mM phosphodiesterase inhibitor, isobutylmethyl xanthine (IBMX, Sigma, St. Louis, MO), and 10 µM adenylate cyclase activator, forskolin, or the ß-adrenergic agonist, isoproterenol (Sigma). Coexpressed ß-adrenergic receptor was used as an alternative to activate adenylate cyclase in most of the experiments described here. Our previous studies indicated that the use of isoproterenol had no apparent effect on the outcomes of chemical modification and pH titration of substitute cysteines and histidines compared to the results using forskolin to raise the cellular cAMP levels (Smith et al., 2001; Liu et al., 2001).

Single-channel recordings
Single CFTR channels were studied at room temperature, in excised, inside-out patches formed from oocytes after the vitelline membrane had been removed manually. Pipettes were pulled in 4–6 stages from borosilicate glass (Sutter Instruments, Novato, CA), and had resistances averaging 6–10 M when filled with pipette solution. In studies of T338C CFTR, the pipette solution contained 196 mM NMDG-Cl, 2 mM MgCl₂, and 5 mM HEPES HemiNa, pH 7.4; 5 mM MES was used in place of HEPES when a pH of 6 was desired for the pipette solution. Typical seal resistances were in the range of 50–200 G. Channels were activated by PKA (Promega, Madison, WI) after excision into intracellular solution (196 mM NMDG-Cl, 2 mM MgCl₂, 0.5 mM Tris-EGTA, 5 mM HEPES HemiNa, and 1 Mg ATP, pH 7.4). Patch currents were measured with an Axopatch 200A amplifier (Axon Instruments, Union City, CA) under voltage-clamp mode and were recorded at 1 kHz. For subsequent analysis, records were digitally filtered at 50–100 Hz and single channel records were analyzed using the event detection features of Clampfit 9 (Axon Instruments). Some experiments were done in TES buffer and were recorded at 10 kHz on Axopatch 200B onto DAT tape (model DTC-790, Sony, Tokyo, Japan) and subsequently filtered at 100 Hz (four-pole Bessel filter, Warner Instruments, Hamden, CT) and acquired by the computer at 400 Hz using the Fetchex program of pClamp (Axon Instruments) for analysis. In these experiments, pipette solution contained 200 mM NMDG-Cl, 5 mM MgCl₂, and 10 mM TES, adjusted to pH 7.4 with Tris). Channels were either activated on-cell with isoproterenol before excising into intracellular solution (200 mM NMDG-Cl, 1.1 mM MgCl₂, 2 mM Tris-EGTA, 1 Mg ATP, and 10 mM TES, pH 7.3, and 50 U/mL PKA (Promega), or were activated by PKA after excision. The single channel current for T338A CFTR was measured using 150 mM symmetrical [Cl^–]. Single-channel i-V plots were obtained by determining the current amplitudes at V_m ranging from –100 mV to 100 mV at 20 mV intervals.

Open and closed current levels were first identified manually and then by transition analysis using a 50% cutoff between open and closed levels. We used an apparent open probability (P_o*) as a measure of channel gating. We defined P_o* as the ratio of NP_o-for-all-levels divided by N, where NP_o-for-all-levels was obtained after single-channel searching using the event detection features in Clampfit 9 and N is the apparent number of channels in a patch.

Recordings of single, T338C channels in experiments involving covalent modification posed additional challenges for two reasons. First, as indicated above, although variable oxidation states of the cysteine could be mitigated by treatment with reducing agents, we could not eliminate the possibility that in any particular patch there could be CFTR channels that would be insensitive to reducing agents and nonreactive toward MTS compounds. In addition, in some preliminary experiments, it was clear that after exposure to a reducing agent, the cysteine at 338 could spontaneously reoxidize to a variable extent over the time course of a typical experiment, 30–120 min. Finally, preliminary experiments indicated that, in contrast to results obtained previously with R334C CFTR, reaction of T338C channels with MTSET⁺ actually reduced single-channel conductance, rendering the resulting single-channel currents more difficult to discriminate from the small (0.1–0.3 pA) background Cl^– channels sometimes seen in patches from oocytes. In view of these potential ambiguities, the single-channel data from experiments involving covalent modification were analyzed in the following way. Amplitude distributions were computed for each current level under each particular condition (reducing agent pretreated, MTSET⁺ modified, treated with reducing agent after MTSET⁺ modification) using the single-channel event detection feature of Clampfit 9 (Axon Instruments). Only opening amplitudes that appeared in all patches under a particular condition were counted as CFTR, and the data were analyzed so as to enable us to determine if changing the experimental condition resulted in the conversion of one amplitude type to another, i.e., an increase in prevalence of openings of one amplitude and a corresponding decrease in another. This procedure should result in the exclusion of background channels as well as CFTR channels that are in a terminal oxidation state that would be insensitive to either reducing agents or MTS reagents. All recordings involving MTSET⁺ modification were conducted at a pipette pH of 6.0 to maximize the conductance of unmodified channels. This acidic pH is expected to slow the rate of the thiol-disulfide exchange reaction, but not to prevent it (Roberts et al., 1986). Recording conditions were designed to maximize the likelihood of recording T338C single channels in each of three states: unmodified; MTSET⁺ modified; and MTSET⁺ modified, but subsequently exposed to 2-ME to reverse the modification (see text).

Several factors inherent in carrying out covalent modification experiments made it very difficult to accurately quantify channel gating. First, to accurately quantify single-channel amplitudes, the ATP concentration in the solution bathing detached patches was often manipulated to enhance the resolution of individual openings in multichannel recordings. Second, to reduce the disulfide bond between MTSET⁺ and the cysteine thiolate at 338 after modification, it was necessary to apply reducing agents to the cytoplasmic side of the patch, a maneuver that has been shown to alter CFTR gating (Harrington et al., 1999). Third, in some of the experiments, long recordings were necessary (20–60 min) during which channel rundown may occur spontaneously. Therefore, the data presented focused on the change in channel amplitude brought about by covalent modification and its comparison to changes in macroscopic currents.

Reagents
Highly polar derivatives of methanethiolsulfonate reagents, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET⁺) and sodium [2-sulfonatoethyl]methanethiosulfonate (MTSES^–) were obtained from Toronto Research Chemicals (Toronto, Canada). N-ethylmaleimide (NEM) was obtained from Sigma. Reducing agents included 2-mercaptoethanol (2-ME) and dithiothreitol (DTT), were obtained from Sigma. The molecular volumes of MTS reagents were determined using a molecular modeling program (Titan, Wavefunction, Irvine, CA).

Whole-cell data analysis
The apparent pK_a of the conductance was determined by titrating the pH of the bath. The value was estimated by fitting the conductance versus pH relation to a modified Henderson-Hasselbalch equation using the curve-fitting routines in SigmaPlot (SPSS, Chicago, IL) (see Eq. 2). The total conductance due to CFTR, g_Cl, was expressed as the sum of two components, one pH-dependent, g(pH) and the other pH-independent, (Eq. 1),

(1)

(2)

where was operationally defined as the minimum conductance that was measured in the most basic solution in a particular experiment and was defined as the maximum change in conductance due to pH titration.

We interpreted the titration behavior of the conductance due to T338C CFTR as reflecting the pH-dependent change in the time-averaged, partial negative charge on any single thiolate anion. We used a modified Henderson-Hasselbalch equation (Eq. 3) to calculate this partial charge just as one would calculate the fraction of thiol in the ionized form

(3)

where z = 1 for cations and –1 for anions and q can range from either –1 to 0 or 0 to 1, depending on the titratable group.

For ease of comparison, in T338C/R334X (X = A or E) CFTRs and T338H/R334C CFTRs in which the cysteine was always blocked by reaction with MTS reagents or NEM, the titration curves were expressed in a normalized form. g(pH) and were expressed as the fractional increase with respect to (Eq. 4),

(4)

Modeling
The effects of charge changes on anion conduction at position 338 were interpreted using two types of models, similar to those employed by Smith et al. (2001). In both models, the pore was viewed as comprising a rate-limiting central region flanked by two vestibules (Dani, 1986; MacKinnon et al., 1989; Lu and MacKinnon, 1994), where the electrical potential created by charged groups can influence local anion distribution. In one model the properties of the rate-limiting region were described using a constant-field, electrodiffusion model (Goldman, 1943) modified to incorporate charged inner and outer vestibules where ion concentrations are in equilibrium with those in the bulk solutions adjacent to the vestibules (Eq. 5). The electrostatic potentials of the inner and outer vestibules were represented by average values, _i and _o, respectively, which were positive for a positively charged vestibule and negative for a negatively charged vestibule. The model was fitted to the data using the curve-fitting routines contained in SigmaPlot (SPSS),

(5)

where V_m is the measured transmembrane potential and P_Cl is the chloride permeability of the narrow region of the pore, [Cl]_b and [Cl]_c are bath and cytoplasmic chloride concentrations, and F, R, and T have their usual meanings. In the second model the rate-limiting region was described using a four-barrier, three-well rate theory scheme incorporating a variable surface potential in the outer vestibule. I-V plots were simulated using a program developed by Dr. Ted Begenisich (Begenisich and Cahalan, 1980) and curves were fitted to the data by eye.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
The conductance of oocytes expressing T338C CFTR is highly sensitive to changes in bath pH
Testing the hypothesis that a cysteine at position 338 resides within the conduction pathway required that we alter the charge at this locus. In previous experiments using R334C CFTR we found that charge change could be effected by means of covalent modification with thiol-directed reagents like MTSET⁺ or MTSES^–, or by using changes in bath pH to alter the partial negative charge on the thiolate anion (Smith et al., 2001). Because the effect of amino acid substitutions at 338 on CFTR conduction had been interpreted as suggesting that T338 might reside near the narrow part of the pore (Linsdell et al., 1997, 1998, 2000; McCarty and Zhang, 2001), we used pH titration to evaluate the effects of charge changes at position 338, hoping to avoid potential obstruction of the pore by MTS reagents. Fig. 2 A contains a plot of g_Cl at V_m = E_rev versus time from a representative experiment (n = 5) designed to test the pH-sensitivity of T338C CFTR. Channels were activated by exposure to a stimulatory cocktail containing 10 µM Isop and 1 mM IBMX (Isop+IBMX). For reasons discussed in Materials and Methods and in a separate communication (Liu et al., 2002), oocytes were first exposed to a reducing agent (0.1–1 mM 2-ME or DTT) for 2–5 min after activation, to favor the simple thiolate form of the engineered cysteine. Afterwards, the bath pH was stepped to pH 6 and then pH 9. Acidifying the bath pH from 7.4 to 6 increased g_Cl at V_m = E_rev from 71.5 ± 10.1 µS to 134.3 ± 12.2 µS (n = 5). Alkalinizing the bath pH from 6 to 9 decreased g_Cl at V_m = E_rev from 134.3 ± 12.2 µS to 21.4 ± 5.2 µS (n = 5). Fig. 2 B contains the I-V plots obtained at pH 9, 7.4, and 6 from the same oocyte. It can be seen that as the bath pH became more acidic the I-V plot changed in two ways: g_Cl at V_m = E_rev increased and the shape changed from inward rectifying toward linearity. As documented in detail in Smith et al. (2001), this result is consistent with the expected influence of a negative charge in the conduction path that is increased at alkaline pH and reduced at a more acidic pH.

View larger version (24K): [in this window] [in a new window]   FIGURE 2  The pH-induced change in conductance of oocytes expressing T338C CFTR. (A) gCl at Vm = Erev versus time. The channels were activated by a stimulatory cocktail containing 10 µM Isop and 1 mM IBMX (Isop+IBMX, hatched bar above). The crosshairs and circles depict times of measurements. At steady state, oocytes were exposed to 1 mM 2-ME (shaded circles) to ensure that the cysteine thiol was in a simple thiolate form. After a wash, the bath pH was changed sequentially to pH 6 (open circles) and pH 9 (solid circles), then to pH 7.4 again. These maneuvers induced changes in both conductance and the shape of the I-V curve. (B) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338C CFTR. (C) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338A CFTR.

View larger version (10K): [in this window] [in a new window]   FIGURE 3  The pH-sensitivity of T338C CFTR conductance was due to the titration of the cysteine thiol. Oocytes expressing T338C or T338S CFTR were first exposed to a stimulatory cocktail containing 10 µM Isop and 1 mM IBMX at pH 7.4. After activation, oocytes were exposed to reducing agents followed by a wash. Bath pH was then changed to either pH 6 (solid bar) or pH 9 (open bar), then to pH 7.4. In some oocytes, after exposure to reducing agents, oocytes were first exposed to NEM after a wash, then to either pH 6 or pH 9. Premodification by NEM prevented the large pH-induced response seen in T338C CFTR conductance. T338S CFTR exhibited no pH-sensitivity.

View larger version (18K): [in this window] [in a new window]   FIGURE 4  Modeling I-V data using continuum (Goldman) and rate theory models. (A) Using the continuum model, the I-V curve corresponding to pH 6 (open squares) obtained from an oocyte expressing T338C CFTR was first fitted by fixing o at zero, resulting in a PCl of 3.51 x 10–7 cm3/s, a Ci of 42 mM, and a i of 20.9 mV. All of the fitted curves are shown as solid lines. I-V curves corresponding to pH 7.4 (shaded triangles) and pH 9 (solid circles) were subsequently fitted with PCl, Ci, and i held constant. The decreased conductances in pH 7.4 and pH 9 with respect to pH 6 were attributed to a shift of the outer vestibule potential by –27 mV and –124 mV, respectively. (B) When using the rate theory model, the predicted currents were scaled according to the macroscopic conductance (gCl at Vm = Erev) at a pH of 6. (The rate theory fitting procedure differed in one detail from that reported previously. Smith et al., 2001, scaled the currents predicted by the model in each condition to the macroscopic I-V plot for that condition. We have found, however, that more satisfactory fits were obtained if a single scaling factor was used for all three plots. This procedure attributed the effects of MTSET+ and MTSES– on the conductance of R334C CFTR to a change in o of 45 mV and –30 mV, respectively, a prediction somewhat different from that reported by Smith et al., 2001, in which the effects of MTSET+ and MTSES– were attributed to a change in o of 50 mV and –10 mV, respectively.) The I-V data obtained at pH 6 (open squares) were fitted using barrier heights (in units of RT) of 4.3, 5, 3, and 5, and well depths (RT) of –1.8, –2, and –1, whereas i and o were fixed at zero. With the same barrier heights and well depths, the I-V data at pH 7.4 (shaded triangles) were fitted by adding a o of –35 mV and the I-V data pH 9 (solid circles) were fitted by adding a o of –95 mV, the default limit of the program.

View larger version (32K): [in this window] [in a new window]   FIGURE 5  The pH-dependence of single-channel amplitudes. (A) Examples of single-channel current records obtained from inside-out patches detached from oocytes expressing T338C CFTR. (B) The i-V plot for T338C CFTR. The fitted curves (solid lines, Goldman equation) were obtained by fixing o and i at zero and Ci at 200 mM, then first fitting the i-V plot for pH 6 (solid circles) to Eq. 4, resulting in a PCl of 1.04 x 10–14 cm3/s. The i-V curve corresponding to pH 7.4 (open circles) was then subsequently fitted while holding PCl constant, resulting in a o of –41 mV. (C) Fitting with the rate theory model. The fitted curves are shown as solid lines. The i-V data at pH 6 were first fit by holding i and o at zero and using barrier heights (RT) of 10.9, 11.5, 9.5, and 12, and well depths (RT) of –1.8, –2, and –1. With the same barrier heights and well depths, the i-V curve at pH 7.4 was fit by adding a o of –50 mV. (Inset) T338A CFTR conductance was insensitive to changes in bath pH. Shown are examples of single-channel i-V plots obtained from inside-out patches detached from oocytes expressing T338A CFTR at pH 6 (solid circles) and pH 7.4 (shaded triangles). All the recordings for T338A CFTR were done in the presence of 150 mM symmetrical [Cl–].

It is also of interest to note that the ratio of the single channel conductance at pH 6 and pH 7.4 for T338C CFTR was 1.8, a value comparable to the ratio observed for the macroscopic conductances (1.7). This result is consistent with the idea that the pH-induced change in the macroscopic conductance was primarily due to a change in single-channel conductance, rather than an effect on channel gating. Because we employed different ionic conditions for single-channel and macroscopic measurements of the conductance ratio (see Materials and Methods), we used the fitted parameters for the single-channel results to determine how this ratio would be affected by changing the ionic conditions. Changing the concentration from 200 mM symmetrical Cl^– (used for single-channel recordings) to that obtained from fitting the macroscopic conductance ([Cl^–]_o= 105.6 mM and [Cl^–]_i = 42 mM) predicted a change in the conductance ratio, _pH6:_pH7.4 of, at most, 9%.

Alteration of the charge on the engineered cysteine thiolate did not affect channel gating. As summarized in Fig. 6 we could discern no relationship between external (pipette) pH and the apparent open probability, P_o*, determined as NP_o/N. At [ATP] = 1 mM, P_o* averaged 0.22 ± 0.06 at pH 6 and 0.21 ± 0.04 at pH 7.4. At [ATP] = 2 mM, P_o* averaged 0.27 ± 0.06 at pH 6 and 0.27 ± 0.02 at pH 7.4. No pH-induced change in P_o* was observed in T338A CFTR, which averaged 0.85 ± 1.2 at pH 6 and averaged 0.74 ± 0.15 at pH 7.4 ([ATP] = 1 mM). We did not study in detail the gating difference between these two mutants, but the result is not surprising in light of previous studies in which amino acid substitutions in the TMs were found to alter CFTR gating (Sheppard et al., 1993; Cotten and Welsh, 1999; Zhang et al., 2000, 2002).

View larger version (11K): [in this window] [in a new window]   FIGURE 6  Alteration of charge at the engineered cysteine at position 338 did not affect channel gating. No relationship exists between external (pipette) pH and the apparent open probability, Po*. , Individual experiments, with either 1 or 2 mM ATP in the intracellular bath solution in which values of Po* were computed (see Methods) over periods 1 min (1 mM ATP) or from 2.5 to 25 min (2 mM ATP). , Mean ± SE for each condition (n = 3–5).

View larger version (15K): [in this window] [in a new window]   FIGURE 7  Titration of conductance due to T338C CFTR. A representative titration curve obtained from an oocyte expressing T338C CFTR. The oocytes used in this group of experiments were treated with reducing agents (2-ME or DTT) at various concentrations (50 µM to 1 mM), followed by pH titration (pH 5.3–10.5) in the presence of the respective reducing agent. The actual data are shown in solid circles. The solid line is the fitted curve. The dashed line is the predicted titration curve for a thiol group having a pKa of 8.3. The dotted line is the predicted titration curve for a thiol group having a pKa of 10.3.

View larger version (16K): [in this window] [in a new window]   FIGURE 8  The pH-induced changes in the conductances of oocytes expressing T338C/R334A, T338C/R334E, T338H, or T338H/R334C CFTR. (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/R334E CFTRs (shaded triangles). The titration was performed in the presence of reducing agents as described in Fig. 7 (n = 5 for each mutant). The solid lines are the fitted curves. (B) Sample titration curves for T338H (open triangle) and T338H/R334C CFTR modified by MTSET+ (solid circles), MTSES– (open circles), and NEM (shaded triangles). The solid lines are fitted curves. The dashed lines are extended curves at low pH values that were calculated based on the fitting results.

Rates of covalent modification of T338C CFTR by MTSET⁺ and MTSES^– are consistent with a model featuring a positive vestibule potential
If, as suggested by the shift in the apparent pK_a of a cysteine or a histidine at 338, this portion of the CFTR pore is characterized by a positive electrostatic potential, the potential should also affect the relative rates of covalent modification by MTSET⁺ and MTSES^– (Stauffer and Karlin, 1994; Pascual and Karlin, 1998; Karlin and Akabas, 1998). Fig. 9 contains the results of experiments in which the time course of modification was measured at pH 7.4 for oocytes expressing either T338C CFTR or a double mutant in which the arginine at 334 was replaced by aspartic acid (T338C/R334D CFTR). Note that covalent modification of T338C CFTR with either reagent decreased the macroscopic conductance. In the case of MTSES^– this decrease seems likely to be due, at least in part, to the deposition of a negative charge in the conduction path, as seen with titration of the cysteine thiolate. The fact that modification by MTSET⁺ also reduced the conductance suggests an additional effect, however, such as a change in gating, partial obstruction of the pore, or both. Single-channel recordings (see below) showed that modification of T338C CFTR by MTSET⁺ reduced single-channel conductance, as expected if the ethyl(trimethylammonium) moiety partially obstructs anion flow through the pore.

View larger version (15K): [in this window] [in a new window]   FIGURE 9  Representative time course of modification of T338C CFTR and T338C/R334D CFTR conductance by MTSET+ (n = 3) and MTSES– (n = 3). Data were normalized according to gCl at Vm = Erev at time zero (go). (A) After activation, oocytes expressing T338C CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 µM MTSET+ () or MTSES– (). (B) After activation, oocytes expressing T338C/R334D CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 µM MTSET+ () or MTSES– (•).

Covalent modification of T338C CFTR by MTSET⁺ adds a positive charge, but also partially obstructs the pore
The reduction of T338C CFTR macroscopic conductance by MTSET⁺ suggested that at this locus, the deposition of the ethyl(trimethylammonium) moiety might partially block the pore. Direct evidence for pore obstruction by MTSET⁺ was obtained by recording T338C CFTR single-channel currents before and after modification. To favor the unmodified condition, oocytes were preincubated in 1 mM 2-ME or DTT for 1 h before recording, and 2-ME (1 mM) was also included in the pipette solution. The first current trace depicted in Fig. 10 A contains a segment of a representative single-channel current trace from an oocyte pretreated with 1 mM DTT for 1 h. 2-ME (1 mM) was also included in the pipette. The majority of current amplitudes observed in this condition were 0.9 pA (V_m = –100 mV), similar to those shown in Fig. 5 A (pH 6, V_m = –100 mV).

View larger version (25K): [in this window] [in a new window]   FIGURE 10  MTSET+ modification of T338C CFTR single-channel conductance. (A) Segments of representative single-channel current traces obtained from an oocyte pretreated with 1 mM DTT for 1 h. 2-ME (1 mM) was also included in the pipette (Patch 1, DTT). An oocyte that was first treated with 1 mM DTT for 5 min, then with 2 mM MTSET+ for 10 min (Patch 2, MTSET+), and from the same patch as in Patch 2, MTSET+, but recorded after 45-min exposure to 20 mM intracellular 2-ME (Patch 2, 2-ME, post-MTSET+). The bath concentration of ATP was manipulated to facilitate the resolution of single-channel events. (B) MTSET+-induced changes in two major current levels observed in three cases: patches obtained from oocytes pretreated with 1 mM ME or DTT for 1 h before patch formation; patches obtained from oocytes pretreated with 2 mM MTSET+ before patch formation with or without MTSET+ included in the pipette solution; and patches containing MTSET+-modified channels that were exposed to 20 mM intracellular 2-ME for >20 min. Although we observed multilevel openings in most of the patches, under reducing conditions the dominant current level was 0.9 pA. MTSET+-treatment caused a marked increase in the frequency of events at the 0.2-pA level and a decrease in the frequency of events at the 0.9-pA level, whereas treatment with 2-ME reduced the frequency of events at the 0.2-pA level and increased the frequency of events at the 0.9-pA level. The results suggested a 75% decrease in single channel current at Vm = –100 mV at pH 6 due to MTSET+-induced modification of T338C CFTR. Ni is the number of the number of single-channel events of each current amplitude, 0.2 pA or 0.9 pA, recorded at Vm = –100 mV, and N is the total number of such events.

In a few experiments, it was possible to record the activity of modified channels and then reverse the modification by perfusing the bath (cytoplasmic side) with membrane permeant reducing agent, 2-ME (20 mM) (Rojas et al., 1991). The third trace depicted in Fig. 10 A was from the same patch as in Patch 2, MTSET⁺, but was recorded after 45-min exposure to 20 mM intracellular 2-ME. The majority of current amplitudes were 0.9 pA after 2-ME exposure, consistent with the notion that modified channels (0.2 pA) were converted to unmodified channels (0.9 pA) by 2-ME. The design of these experiments precluded a comparison of single-channel gating in the different conditions as the bath concentration of ATP was manipulated to facilitate the resolution of single-channel events, and incubation of detached-patches with 2-ME might be expected to influence gating (Harrington et al., 1999).

The fractional distribution of single-channel current amplitudes for the three conditions obtained from multiple patches is shown in Fig. 10 B. After extensive incubation in 2-ME, the single-channel current amplitudes were dominated by a current of 0.9 pA, identical to the current amplitude at pH 6 shown in Fig. 5 A. There were a small number (<10%) of openings at a level of 0.2 pA. These currents cannot represent the MTSET⁺-modified channels, as these oocytes had never been exposed to the compound. After prolonged exposure to MTSET⁺ the dominant single-channel current amplitude was 0.2 pA and the overall occurrence of 0.9 pA current was reduced to 20%. Approximately one-third of the patches had no 0.9 pA currents in this condition. Exposure of modified patches to 20 mM 2-ME resulted in a reappearance of the 0.9-pA openings together with a small number of 0.2-pA events. This result is consistent with the notion that the 0.2-pA event represented MTSET⁺-modified T338C CFTR channels. Based on the single-channel amplitude observed under reducing conditions, it seems likely that the 0.2-pA events included a small contribution from a background channel or a terminal oxidation state of T338C CFTR; see Materials and Methods. The fact that the modification of the T338C CFTR channel was not always complete may reflect the acidic pH as well as some degradation of the MTSET⁺. The reduction in single-channel conductance of 75% was comparable to that seen in macroscopic recordings (see below) in which the reduction in conductance varied from 20 to 80% (Fig. 12), suggesting that the attenuation of macroscopic conductance is largely attributable to a reduction in single-channel conductance. The variability in the impact of MTSET⁺ on macroscopic conductance may indicate that not all the cysteines were in the simple thiolate form, which is required for MTSET⁺ reaction. This variability persisted even after overnight treatment of oocytes with 1 mM DTT (X. Liu and D. C. Dawson, unpublished observations), suggesting that in some cases the cysteine at 338 was oxidized to either a sulfinic acid or sulfonic acid that was insensitive to reducing agents.

View larger version (17K): [in this window] [in a new window]   FIGURE 12  The pH-dependent effect of MTSET+ on whole-cell T338C CFTR conductance. After activation, oocytes were first exposed to 1 mM 2-ME or DTT, then to either pH 6 or pH 9, followed by exposure to MTSET+ (1 mM) at the same pH. (A) At pH 6, MTSET+ reduced the conductance due to T338C CFTR by variable amount (20–80%). (B) At pH 9, the conductance was increased by MTSET+.

View larger version (15K): [in this window] [in a new window]   FIGURE 11  Real-time modification of T338C-CFTR channels activated by PKA and ATP in an excised, inside-out patch. The pipette was backfilled with pipette solution containing 100 µM MTSET+. During the course of the recording, MTSET+ diffused to the pipette tip, and modified the T338C-CFTR channels there. Shown are several segments of record from an experiment lasting >25 min; the elapsed time from forming the seal to the beginning of the segment is shown below the trace. Current amplitudes of representative openings are listed. Pipette solution was pH 6.0 to enhance channel amplitudes before modification. As is often the case in long experiments of wt CFTR expressed in oocytes, channel activity ran down during the course of the record, so the effect of MTSET+ on gating could not be readily quantified.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY MATERIAL ACKNOWLEDGEMENTS REFERENCES

 
T338 lies within the CFTR pore
The results presented here are consistent with the hypothesis that T338 lies within the anion conduction pore of CFTR, but the location of this residue has been a matter of some controversy. Cheung and Akabas (1996) reported that T338C CFTR did not react with externally applied MTSET⁺ or MTSES^– and concluded that the residue did not lie on the outward-facing, water-accessible surface of the protein. The effects of amino acid substitution at this locus, however, led Linsdell et al. (1997, 1998, 2000) and McCarty and Zhang (2001) to identify this residue as critical for anion permeation and binding. Although the reason for the discrepancy between our findings and those of Cheung and Akabas (1996) is not completely clear at present, one contributing factor may be the increased reactivity (reduced pK_a) of the cysteine thiol at position 338 which renders it more susceptible to reactions that could change its oxidation state and effectively block reactions with MTS reagents.

Two results presented here argue that T338 lies within the pore. First, a cysteine substituted at 338 was clearly accessible to polar thiol-directed reagents as well as protons and the polar reducing agent, dithiothreitol. Second, charge changes at 338, effected by pH titration of the cysteine thiolate, altered anion conduction in the manner predicted by a model featuring a charge located in the outer vestibule of the pore. Thus this locus meets the criterion proposed in a previous study of residue 334 (Smith et al., 2001) for a pore-lining residue, namely that altering the charge in the vestibule of the pore should change anion conduction in a charge-dependent manner, the key criterion being a simultaneous alteration of conductance and the shape of the I-V (or i-V) plot. Although this criterion may not apply to all pore-lining residues, it provides one test for residues that lie in the outer vestibule of the pore, defined generally as a wider region that precedes a narrow, rate-limiting portion of the pore where anions bind (Cai and Jordan, 1990; Dani, 1986; MacKinnon et al., 1989). The observation that modification of T338C CFTR by MTSET⁺ produced a net reduction in single-channel conductance, whereas similar modification of R334C CFTR increased single-channel conductance, is compatible with the notion that the pore narrows over the length of the helical turn.

The pK_a of T338C CFTR is consistent with a model in which T338C resides in the pore where it senses a positive electrostatic potential
The pH titration of the conductances of oocytes expressing T338C CFTR was consistent with the stabilizing effect of a nearby positive charge on the thiolate anion, and the behavior of double mutants suggested that one source of this charge could be R334. The effects of substitution for R334 were not completely consistent, however. Whereas the alanine substitution rendered the apparent pK_a more than one pH unit more alkaline, neither the glutamic acid nor aspartic acid substitution produced a significant additional shift. This apparent asymmetry was evident in both of the independent methods used to estimate _o, i.e., the pK_a and the ratio of reaction rates for MTSET⁺ and MTSES^–. Although the underlining cause of the asymmetry is unclear at present, the similarity of the results of the two independent methods for determining _o nevertheless provides strong evidence that a positive electrostatic potential exists in the vestibule. The asymmetry suggests that the insertion of the negative charge (Glu or Asp) might change the conformation of the protein in such a way as to diminish the impact of the charge on _o, or increase the impact of other, as yet unidentified, positive charges.

The influence of the local protein environment on the pK_a of a titratable group has been of interest to investigators for many years and has been modeled for proteins of known structure (Antosiewicz et al., 1994, 1996; Bashford and Karplus, 1990; Bashford and Gerwert, 1992; Dillet et al., 1998; Fitch et al., 2002; Gilson and Honig, 1988; Oberoi and Allewell, 1993; Peters et al., 1998; Yang et al., 1993). In the case of T338C CFTR, although we lack the information as to a three-dimensional structure, we can nevertheless make use of some of the general principles that have emerged from the analysis of other proteins. As shown in Supplementary Material, the change in pK_a of a particular titratable group in the putative vestibule can be approximated by Eq. 6,

(6)

where is the component of the time-averaged electrostatic potential sensed by the titratable group that is attributable to local charge other than that of the titratable group itself; in this case, the cysteine thiolate anion. Ideally, would represent the potential that pertains, in our model, to the vestibule of the wt CFTR pore lacking the engineered cysteine. We cannot expect this to be exactly the case, however, due to the inevitable effects of amino acid substitution on protein conformation. The total potential, for T338C CFTR, would also contain a pH-dependent component due to the cysteine thiolate at 338. As indicated above, at alkaline pH, the charge at 338 would be likely to make a large contribution to in T338C CFTR.

Eq. 6 is based on two simplifying assumptions (see Supplementary Material). First we assumed that the change in pK_a due to differential solvation of the protonated form of the titratable group is negligible (Sitkoff et al., 1994; Florian and Warshel, 1997); and second, we assumed that the change in pK_a due to differential solvation of the thiolate anion is zero, as if the anion is solvated in the vestibule as in free solution. By setting the solvation penalty to zero we ensure that Eq. 6 provides an estimate of the minimum value of Any solvation effect, as it would shift the pK_a of the thiolate in alkaline direction, would increase the positive value of required to achieve the observed pK_a of the cysteine at 338.

There are at least two estimates of the vestibule potential seen by a cysteine at 338, which are of interest. One is the absolute value of with respect to the bath that would be derived from the shift in pK_a with respect to that for a simple thiol in aqueous solution. A second calculation would be the component of due to R334, A calculation of the absolute value of relative to the bath is complicated by some ambiguity as to what to take as the pK_a of the cysteine thiolate in the aqueous solution. Choices would include free cysteine, or simple compounds such as methylthiol or ethylthiol or linear helical peptides containing a cysteine (Irving et al., 1964; Kreevoy et al., 1964; Stewart, 1985; Kortemme and Creighton, 1995). The pK_a values range from 8.3 for free cysteine to 10.3 for methylthiol or ethylthiol, and would translate into values for ranging from 53 mV to 171 mV positive with respect to the bath using a pK_a of 7.4 for T338C CFTR. It is important to note in this regard that conduction models—from Goldman and rate theory—used to analyze the change in I-V plots induced by change in bath pH yield only relative values of Although arguments might be advanced in favor of choosing one of these pK_a values over another, we chose to focus on the electrostatic effect of R334.

A comparison of the apparent pK_a of T338C CFTR with that of the double mutant, T338C/R334A, suggests that the amino acid substitution at position 334 shifted the pK_a from 7.4 to 8.8 or 1.4 units. This pK_a implies a change in (Eq. 6) of 83 mV. A comparison of T338C/R334A (pK_a = 8.8) with T338C/R334E (pK_a = 8.9), would suggest a further change in of –6 mV.

The relative rates of modification of T338C CFTR by MTSET⁺ and MTSES^– provide another estimate of (Stauffer and Karlin, 1994; Pascual and Karlin, 1998; Karlin and Akabas, 1998; Wilson et al., 2000; Elinder et al., 2001). To analyze the rates of modification we assumed that the concentration of the MTS reagents in the vestibule is determined by as if the reagents equilibrate with the component of due to local charge other than that of the cysteine thiol. The ratio of the rates of modification is given by Eq. 7 (see Supplementary Material),

(7)

Using the ratio for of 1/12 measured for 2-ME (Karlin and Akabas, 1998), the relative rates of modification for these two compounds yields a value for of 54 mV for T338C CFTR and –4 mV for T338C/R334D CFTR, comparable to the values estimated from the shift in cysteine pK_a resulting from the replacement of arginine by alanine or aspartic acid. The absolute values of must be interpreted with caution since we do not know to what extent structural differences between these two mutants might have resulted from the amino acid substitution (see below), but both of the measurements used to estimate the electrostatic potential of the vestibule indicated an asymmetry between the impact of basic and acidic residues at 334.

Prediction of the electrostatic effects of R334
If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in calculated from the difference in the pK_a of T338C/R334 and T338C/R334A can be taken to be a crude measure of the component of