CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore

Abstract

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 gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa 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 pKa 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.

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.

FIGURE 2

The pH-induced change in conductance of oocytes expressing T338C CFTR. (A) g_Cl at V_m = E_rev 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.

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.

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 Ψ_o at zero, resulting in a P_Cl of 3.51 × 10⁻⁷ cm³/s, a C_i of 42 mM, and a Ψ_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 P_Cl, C_i, and Ψ_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 (g_Cl at V_m = E_rev) 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 Ψ_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 Ψ_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 Ψ_i and Ψ_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 Ψ_o of −35 mV and the I-V data pH 9 (solid circles) were fitted by adding a Ψ_o of −95 mV, the default limit of the program.

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 Ψ_o and Ψ_i at zero and C_i at 200 mM, then first fitting the i-V plot for pH 6 (solid circles) to Eq. 4, resulting in a P_Cl of 1.04 × 10⁻¹⁴ cm³/s. The i-V curve corresponding to pH 7.4 (open circles) was then subsequently fitted while holding P_Cl constant, resulting in a Ψ_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 Ψ_i and Ψ_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 Ψ_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⁻].

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, P_o*. ○, Individual experiments, with either 1 or 2 mM ATP in the intracellular bath solution in which values of P_o* 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).

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 pK_a of 8.3. The dotted line is the predicted titration curve for a thiol group having a pK_a of 10.3.

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.

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 g_Cl at V_m = E_rev at time zero (g_o). (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⁻ (•).

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 V_m = −100 mV at pH 6 due to MTSET⁺-induced modification of T338C CFTR. N_i is the number of the number of single-channel events of each current amplitude, 0.2 pA or 0.9 pA, recorded at V_m = −100 mV, and N is the total number of such events.

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.

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⁺.