Relative movements of transmembrane regions at the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore during channel gating

Abstract

Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.

FIGURE 1.

Spontaneous and oxidant-induced disulfide bond formation between cysteine side chains in TM6 and TM11. A and B, example whole cell current recordings for R334C/G1127C during voltage steps (−80 mV to +80 mV in 20-mV increments from a holding potential of 0 mV). Currents were recorded after attainment of a stable whole cell configuration (control), after application of cAMP stimulatory mixture (see “Experimental Procedures”) (+cAMP), after application of DTT (A) or CuPhe (B), and after CFTR channel inhibition by addition of 50 μm GlyH-101. Current-voltage relationships from these two cells, measured from the final 100 ms of the voltage steps for each of the four sets of current traces shown, are illustrated to the right. C and D, mean effect of DTT (C) or CuPhe (D) application on whole cell current amplitude for each of the 15 double cysteine mutants studied. In both cases, the TM6 residue substituted by cysteine (Arg-334, Lys-335, or Thr-338) is named above the panel, and the TM11-ECL6-TM12 residue substituted is below the panel. In both C and D, the mutants in which treatment with DTT or CuPhe resulted in a significant change in current amplitude (p < 0.05) are represented by black bars, and those mutants for which there was no significant change are represented by gray bars. The means of data from three to six cells are shown in C and D.

FIGURE 2.

Irreversibility of CuPhe inhibition in double cysteine mutants. A, example whole cell currents recorded at +30 mV for T338C/S1118C from cells that had been pretreated with CuPhe either together with FSK (left panel) or without FSK (right panel) for 5 min. Following extensive washing and attainment of the whole cell patch configuration, channel activation by application of cAMP stimulatory mixture revealed currents that were increased by DTT application (5 mm) only if CuPhe was applied together with FSK. In each case, the identity of CFTR currents was confirmed using the specific inhibitor GlyH-101 (50 μm). B, mean effect of DTT application on whole cell current amplitude for the three double cysteine mutants named, following different pretreatment conditions. Asterisks indicate a significant difference from no pretreatment conditions (p < 0.01), and daggers indicate a significant difference from pretreatment with CuPhe in the absence of FSK (p < 0.01). The means of data from three to six cells are shown. C, stability of CuPhe inhibition of R334C/G1127C and reversibility by excess DTT. Example whole cell current from a cell treated sequentially with cAMP stimulatory mixture, CuPhe, DTT, and GlyH-101.

FIGURE 3.

Cadmium inhibition of single and double cysteine mutants. A–F, example whole cell currents recorded at +30 mV for Cys-less (A), R334C (B), R334C/T1122C (C and F), and R334C/G1127C (D and E). CFTR currents were activated by application of cAMP stimulatory mixture in each case, prior to the addition of Cd²⁺ to the extracellular solution at the concentrations illustrated. The cells were pretreated with DTT (5 mm) except where stated otherwise (E and F). G, mean inhibitory effect of Cd²⁺ on different channel variants under different conditions as illustrated. Asterisks indicate a significant difference from the same channel variant without pretreatment (p < 0.0001), and daggers indicate a significant difference from the corresponding single TM6 mutant (R334C or T338C) under the same pretreatment conditions (p < 0.005). Note that CuPhe-induced disulfide formation prevented strong inhibition by Cd²⁺ ions in all double cysteine mutants tested. The means of data from three to five cells are shown. Ctrl, control.

FIGURE 4.

Disulfide bond formation in constitutively open channels. A, example whole cell current recording and corresponding current-voltage relationship for R334C/G1127C/E1371Q, illustrating a lack of inhibition by CuPhe. Compare with R334C/G1127C in Fig. 1 under identical experimental conditions. Note the overlapping current-voltage relationships for control (●), +cAMP (○), and +CuPhe (▾). B, mean effect of CuPhe treatment on whole cell current amplitude for the three CuPhe-sensitive double mutants named, with either the native Glu present at position 1371 (1371E, white bars) or with Gln substituted at this position (1371Q, black bars). Asterisks indicate a significant difference from the effect in a 1371E background. *, p < 0.05; **, p < 0.00001. The means of data from three to six cells are shown. Ctrl, control.

FIGURE 5.

Cadmium inhibition in constitutively open channels. A, example whole cell currents recorded at +30 mV for T338C/S1118C (left panel) and T338C/S1118C/E1371Q (right panel), showing the increased sensitivity to inhibition by external Cd²⁺ (10 μm) in E1371Q-containing channels. B, effect of the E1371Q mutation on Cd²⁺ sensitivity in single and double cysteine mutants. Cadmium concentrations, chosen to give ∼50% inhibition for each mutant, are given below the name of the mutant used. Asterisks indicate a significant difference from the effect in a Glu-1371 background (p < 0.05). The means of data from three to four cells are shown. Ctrl, control.

FIGURE 6.

Evidence for spontaneous disulfide bond formation in constitutively open channels. A, example whole cell currents recorded at +30 mV for R334C/T1122C/E1371Q, showing that inhibition by external Cd²⁺ (3 μm) is dependent on prior treatment with DTT to reduce spontaneous disulfide bonds. B, dependence of Cd²⁺ inhibition on different pretreatment conditions in different cysteine double mutants in an E1371Q background. Cadmium concentrations, chosen to give ∼50% inhibition for each mutant, are given below the name of the mutant used. Asterisks indicate a significant difference from the effect without pretreatment (p < 0.0005). The means of data from three to six cells are shown. Note that the Cd²⁺ sensitivity of the single cysteine mutants R334C/E1371Q and T338C/E1371Q was independent of DTT pretreatment (not shown), as shown in Fig. 3 for R334C and T338C. Ctrl, control.

FIGURE 7.

Proposed alignment and relative movement of TMs 6 and 11 during channel opening and closing. A, two-dimensional location of mutated residues in TM6 (left panel) and TM11-ECL6-TM12 (right panel). For TM6, side chains accessible to the extracellular solution are to the right, and nonaccessible side chains are to the left (21, 23, 29, 40). For TM11–12, accessible side chains are outside the loop, and nonaccessible side chains are inside (15, 25). Residues mutated in the present study are in red. Functional evidence suggests that other accessible TM11–12 residues Val-1129, Ile-1131, and Ile-1132 are located at more superficial positions in the pore outer mouth (15). B and C, proposed location of disulfide bonds formed in closed (B) and open (C) channels and inferred relative alignment of TMs 6 and 11 in these two states (Table 1). The suggested relative movement of these two TMs is indicated by the red arrows in C; when the channel opens, TM6 moves relatively “downward” (toward the cytoplasm) and/or TM11 moves relatively “upward” (toward the extracellular solution).