Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule

Abstract

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

Figure 1.

Candidate pore-lining secondary structures. (A) Structure of ClC-ec1 (1OTS) as viewed from within the membrane. The subunits are colored gray and cyan, with secondary structures lining the intracellular vestibule of the gray subunit colored as follows: helix C and loop CD, orange; helix D and loop DE, magenta; helix E, light pink; helix F, periwinkle; helix J, royal blue; helix M, tan; helix R, red. The figure was created using Pymol (DeLano, 2002). (B) Alignment of secondary structures screened. ClC-ec1 and ClC-0 sequences were aligned using Clustal-W, with slight manual adjustment. Conserved residues are shaded. The regions screened are indicated by bars above the ClC-0 sequence. The beginning of helix J is omitted.

Figure 2.

Changes in macroscopic open-channel current correspond to changes in single-channel current. Protocol A (A) and K519C current response (B) before (top left) and after (top middle) reaction with MTSEA, and after reversal with DTT (top right). The dashed line indicates zero current. The open-channel current (bottom left) and apparent open probability (bottom right) before (open circles) and after (filled circles) reaction with MTSEA, and after DTT (crosses) (derived as described in materials and methods). (C) I515C current before (top left) and after (top middle) reaction with MTSES. Top right shows current after reaction with MTSES with the y axis adjusted for ease in viewing. The bottom panels show open-channel current (bottom left and middle, same data on two scales) and voltage-dependent gating (bottom right) before (filled circles) and after (open circles) reaction with MTSES.

Figure 3.

MTSES sensitivity of candidate pore-lining residues. Change in open-channel current −100 mV from the reversal potential, before (I_before) and after (I_after) application of MTSES shown as percent decrease in current {100*((I_before − I_after)/I_before)}. Bars show mean ± SEM, number of patches in parentheses. For residues for which the decrease in current was distinguishable from drift, the percent decrease is reported for reactions that had reached >95% completion. Since S123C passes very little current at negative voltages, the decrease in current at +40 mV from the reversal potential is shown.

Figure 4.

Time courses and rates of MTSES reactions. Protocol B was used while MTSES was flowed over the cytoplasmic side of the channels. (A and B) The open-channel current at −150 mV is plotted versus time, with arrows indicating when MTSES was introduced, and the traces fit to single exponentials. (A) Mutants that reacted faster than 0.015 s⁻¹ (150 M⁻¹s⁻¹). (B) The three MTSES-sensitive mutants that reacted more slowly than 0.015 s⁻¹, with only every 10th point plotted for clarity. For S123C, the open probability is very low at −150 mV, so the rate was based on the decrease in current at +50 mV. (C) Rates of reaction with 100 μM MTSES (mean ± SEM, with number patches in parentheses) as determined from fits such as those shown in A and B. For a second order reaction under these conditions, a rate of 0.1 s⁻¹ corresponds to a rate constant of 10³ M⁻¹s⁻¹. Asterisks indicate mutants that were further characterized (those that react faster than 0.015 s⁻¹ and in which current decreased >35%), Fig. 6.

Figure 5.

Effects of MTSES on open-channel current and voltage-dependent gating. Two examples of MTSES-sensitive mutants: I308C on helix J (A) and G122C on loop CD (B). Current responses to protocol A are shown before (top left) and after (top middle) exposure to MTSES. Top right shows the same data as top middle but with the y axis adjusted for ease in viewing. The open-channel current (bottom left, bottom middle with y axis adjusted) and apparent open probability (bottom right) before (filled circles) and after (open circles) reaction with MTSES.

Figure 6.

MTSES-sensitive sites: effects on voltage-dependent gating and rectification. Rectification and voltage-dependent gating parameters (derived as described in materials and methods) before (dark gray) and after (light gray) reaction with MTSES (mean ± SEM, number patches in parentheses). (A) Rectification metric; (B) V _o, the midpoint of the voltage–activation curve; (C) z, the effective gating charge; (D) P _min, the minimum open probability.

Figure 7.

Effects of AMS. (A) Chemical structures of MTSES and AMS adducts after reaction with a cysteine residue. (B–D) AMS effect on M311C. (B) The open-channel current at −150 mV is plotted versus time, with arrow indicating when 100 μM AMS was introduced, and the trace fit to a single exponential. (C) Current response to protocol A, before (left) and after (middle, right) reaction with AMS. (D) Open-channel current (left, middle) and apparent open probability (right), before (filled circles) and after (open circles) treatment with AMS. (E) Percent decrease in open-channel current −100 mV from the reversal potential, calculated as in Fig. 3. Reactions went to >95% completion except for M311C, which reached >91% completion. (F) Rates of reaction with 100 μM AMS as determined from fits such as the one shown in B. WT*, M311C*, and R312C*, shown with black bars, indicate rates of reaction with AMS for patches that had been pretreated with MTSES. (G) Rectification metric (as in Fig. 6 A) before (white bars) and after (dark bars) reaction with AMS. (H and I) Pretreatment with MTSES reduces effects of AMS. (H) Time courses as in B were used to calculate percent decrease in current at −150 mV caused by AMS with (black bars) and without (gray bars) pretreatment with MTSES. Percent decrease was calculated from time courses rather than as in E because pulse protocol A was not used between treatment with MTSES and AMS. (I) Rectification metric (as in Fig. 6 A) for untreated patches (white bars), patches treated with AMS alone (gray bars), MTSES alone (hatched bars) and MTSES followed by AMS (black bars). For R312C, the difference between AMS alone (gray bars) and MTSES followed by AMS (black bars) is significant (P = 0.027, Student's t test). All bar graphs show mean ± SEM, number patches in parentheses.

Figure 8.

Disulfide bond formation between I308C and I515C. (A–C) Effect of copper phenanthroline and subsequent DTT on the I308C-I515C double mutant. (A) Current response to protocol A before (left) and after (middle) reaction with copper phenanthroline, and after DTT (right). (B) Open-channel current (left and middle) and open probability (right), before (open circles) and after (closed circles) copper phenanthroline, and after DTT (crosses). (C) Time courses of open-channel current at −150 mV as in Fig. 4, for the reaction catalyzed by copper phenanthroline (left) and reversed by DTT (right). Arrows indicated the time that the reagents were introduced. The time courses for the reaction induced by copper phenanthroline did not fit a first-order exponential, and the reversal by DTT was so fast as to be limited by solution exchange. (D) Percent decrease in open-channel current after treatment with copper phenanthroline, calculated as in Fig. 3. All patches included were subsequently treated with DTT, which had little effect on the I308C and I515C single mutants but restored the I308C-I515C double mutant current to near its original value. (E) The disulfide bond between I308C and I515C causes outward rectification. Rectification before (dark gray) and after (light gray) treatment with copper phenanthroline. For details on the rectification metric, see materials and methods. All bar graphs show mean ± SEM, number patches in parentheses.

Figure 9.

Residues sensitive to thiol modification mapped onto the ClC-ec1 structure (1OTS). (A) MTSES/AMS sensitivity correlates with predicted secondary structures. Helical wheels for regions predicted to be helix R and helix J. Tested residues are boxed according to their MTSES sensitivity reported in Fig. 3 (red, >65% decrease in current; magenta, 35–45%; blue, <35%), and marked according to their AMS sensitivity as reported in Fig. 7 (stars, >0.01 s⁻¹ rate of AMS inhibition; curls, <0.01 s⁻¹). (B) A view from within the membrane, with the subunits shown in gray and cyan and chloride ions spacefilled in green. In B and C, AMS/MTSES-sensitive residues are spacefilled in yellow, and all other residues tested are colored according to secondary structure (helix C and loop CD, orange; helix D and loop DE, magenta; helix E, light pink; helix F, periwinkle; helix J, royal blue; helix M, tan; helix R, red). (C) Stereoview of one subunit, viewed from inside the cell at an angle so that the bound chloride is visible. All tested residues spacefilled; AMS/MTSES-sensitive residues (yellow) cluster around bound chlorides. (D) Side view as in B, highlighting in magenta the proximity of Q277 (helix J, I308 equivalent) to I448 (helix R, I515 equivalent) in the ClC-ec1 structure. (E) Cytoplasmic view as in C showing spacefilled the AMS-sensitive residues (yellow) and the MTSES-sensitive residues reacting more slowly (periwinkle) or faster than 0.015 s⁻¹ (150 M⁻¹s⁻¹) (magenta for residues with large effects on open-channel rectification, cyan for residues with smaller effects on open-channel rectification).