Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel

Abstract

Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl(-) ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl(-) ion permeation through the pore.

Figure 1.

Location of positively charged amino acid residues within the CFTR TM regions. The CFTR protein includes 12 TM regions, which are aligned as described previously (Riordan et al., 1989; Dawson et al., 1999; McCarty, 2000). Positively charged lysine and arginine residues are shaded. The present study introduced mutations of a number of arginine residues predicted to lie close to the intracellular ends of TM1 (R80), TM4 (R242), TM5 (R303), TM6 (R352), TM8 (R933), and TM11 (R1102) (“Inner Arginines”), as well as more centrally located residues closer to the center of TM2 (R134) and TM10 (R1030) (Central Arginines).

Figure 2.

Mutagenesis of positively charged arginine residues induces CFTR channel current rectification. (A) Example leak subtracted macroscopic I-V relationships from different CFTR channel variants in inside-out membrane patches, after maximal channel activation with ATP, PKA, and PPi. In contrast to wild-type CFTR, mutants R303E and, to a lesser extent, R352E showed outward rectification of the I-V relationship with symmetrical 154 mM Cl⁻ solutions. (B) Mutagenesis-induced rectification was specific for these two positions; charge-changing mutations at four other arginine residues did not significantly alter I-V shape. The degree of I-V rectification is quantified as the rectification ratio, as defined in Materials and methods. Mean of data from three to four patches. * indicates a significant difference from wild type (P < 0.0001, two-tailed t test).

Figure 3.

Charge and chloride dependence of current rectification in mutant forms of CFTR. (A) The degree of rectification depends on the charge of the amino acid side chain present at position 303 or position 352. (B) The degree of rectification in R303E, R303Q, and R352Q is dependent on the Cl⁻ concentration. Rectification was quantified at different symmetrical Cl⁻ concentrations. * indicates a significant difference from the rectification ratio of the same channel variant with 154 mM Cl⁻ (P < 0.05, two-tailed t test). Unfortunately, because of low current density in this mutant, R352E could not be studied at low Cl⁻ concentration. Mean of data from 3–10 patches in both A and B.

Figure 4.

Mutagenesis of positively charged arginine residues reduces CFTR single channel conductance. (A) Example single channel currents recorded from different channel variants at a membrane potential of −30 mV. For each trace, the line to the left represents the current level when the channel is closed. (B and C) Mean single channel current–voltage relationships constructed from such recordings for wild type (filled circles in each case) and channels bearing mutations at either R303 (B) or R352 (C). (D) Mean unitary conductance for wild-type CFTR and for channels bearing charge-changing mutations at each of six “inner arginines.” Unitary conductance was estimated as the slope of individual single channel current–voltage relationships such as those shown in B and C. * indicates a significant difference from wild type (P < 10⁻¹⁰, two-tailed t test). (E) Unitary conductance depends on the charge of the amino acid side chain present at position 303 (•) or position 352 (◯). Mean of data from three to five patches in B–E.

Figure 5.

Mutagenesis of positively charged arginine residues causes rectification of the single channel current–voltage relationship. (A) Example single channel currents carried by Cl⁻ influx or Cl⁻ efflux atthe membrane potentials indicated. For each trace, the line to the left represents the current level when the channel is closed. (B) Mean single channel current–voltage relationships constructed from such recordings for wild type (▪), R303E (•) and R352E (◯). Mean of data from three to five patches.

Figure 6.

Strong chloride concentration dependence of unitary conductance in mutant channels. (A) Example single channel currents recorded from wild type, R303E, and R352E at different intracellular Cl⁻ concentrations (154 or 304 mM, as indicated), at a membrane potential of −20 mV. For each trace, the line to the left represents the current level when the channel is closed. (B) Mean single channel current–voltage relationships constructed from such recordings demonstrate that increasing intracellular Cl⁻ concentration has a greater effect on these two mutants than on wild type. This is further shown from the mean unitary conductances estimated from the slope of individual single channel current–voltage relationships (C). Bars on the left in this panel illustrate the mean conductance estimated under different ionic conditions, while gray bars on the right show the mean percent change in conductance comparing 304 mM with 154 mM Cl⁻. Mean of data from three to five patches in B and C.

Figure 7.

Modification of CFTR by intracellular MTSES. (A) Example leak-subtracted macroscopic I-V relationships in inside-out membrane patches with symmetrical 154 mM Cl⁻ solutions, after maximal channel activation. Currents were recorded at various times before (time zero) and after application of 200 μM MTSES to the intracellular solution. Time after application of MTSES, in minutes, is shown alongside each I-V relationship. (B) MTSES-induced changes in I-V shape. Data from the same patches as in A, recorded before (control) and 6 min after addition of MTSES, are scaled to show the development of outward rectification in R303C but not wild type. (C) Time course of current rundown for these two example patches; current at +80 mV (I ₊₈₀) and −80 mV (I ₋₈₀) was monitored continuously after application of MTSES. (D) Lack of rundown over the same timescale on addition of vehicle alone in wild type. (E) Change in rectification ratio with time after addition of MTSES for the two patches used in A–C.

Figure 8.

Modification of CFTR by intracellular MTSET. (A) Individual examples of the time course of MTSET-induced current rundown in wild type and R303C. As described in Fig. 7 for MTSES, current was monitored at different times after addition of 2 mM MTSET to the intracellular solution. (B) Change in rectification ratio with time after addition of MTSET for these two patches.

Figure 9.

Modification of surface charge by intracellular MTS reagents alters I-V shape. Mean I-V rectification was measured before (−) and 4–6 min after (+) application of either MTSES (200 μM) or MTSET (2 mM) to the intracellular solution. * indicates a significant difference from matched, pre-MTS control values (P < 0.05, paired t test). Mean of data from three to five patches.

Figure 10.

Effect of mutagenesis of “central arginine” residues. (A) Example leak-subtracted macroscopic I-V relationships from R134Q and R1030Q-CFTR in inside-out membrane patches with symmetrical 154 mM Cl⁻ solutions, after maximal channel activation. (B) Quantification of the rectification of the I-V relationship for these two channel mutants at different symmetrical Cl⁻ concentrations. * indicates a significant difference from wild type at the same Cl⁻ concentration (P < 10⁻⁴, two-tailed t test), † indicates a significant difference from R134Q at 154 mM Cl⁻ (P < 0.005, two-tailed t test). Mean of data from three to eight patches. (C) Example single channel currents recorded from these two channel mutants at a membrane potential of −30 mV. For each trace, the line to the left represents the current level when the channel is closed. For R134Q, the tiny current associated with a putative channel opening is identified by a line above it. Note the different scale to the ordinate axis for these two current traces. (D) Mean single channel current–voltage relationships for wild type (filled circles) and R1030Q (open circles). Mean of data from four to five patches.

Figure 11.

Anion selectivity of mutant forms of CFTR. Example leak-subtracted macroscopic I-V relationships from different CFTR channel variants in inside-out membrane patches after replacement of 75% of extracellular NaCl with sucrose. Under these ionic conditions, each of the I-V relationships reverses close to the calculated Cl⁻ equilibrium potential (E_Cl) of +33.4 mV. Examples of similar data obtained from three to four patches for these channel variants and also for R80E, R242E, R352Q, R933E, and R1102E (not depicted).