Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

FIGURE 1.

Rate of modification of T338C and L102C by internal MTSES. A and B, sample time courses of macroscopic currents (measured at −50 mV) carried by different CFTR channel variants as indicated in inside-out membrane patches. Current amplitudes were measured every 6 s following attainment of stable current amplitude after channel activation. In each panel, 200 μm MTSES was applied to the cytoplasmic face of the patch at time zero as indicated by the hatched bars. The decline in current amplitude following MTSES application has been fitted by a single exponential function. C, average modification rate constants (k) for MTSES, calculated from fits to data such as those shown in A and B. Asterisks indicate a significant difference from the cysteine mutants T338C and L102C (black bars) (p < 0.05). Data are mean from three or four patches.

FIGURE 2.

Rate of modification by internal Au(CN)₂⁻. A and B, sample time courses of macroscopic currents carried by different CFTR channel variants as indicated in inside-out membrane patches. In each panel, 2 μm Au(CN)₂⁻ was applied to the cytoplasmic face of the patch at time zero as indicated by the hatched bars. The decline in current amplitude following Au(CN)₂⁻ application has been fitted by a single exponential function. C, average modification rate constants (k) for Au(CN)₂⁻, calculated from fits to data such as those shown in A and B. Asterisks indicate a significant difference from the cysteine mutants T338C and L102C (black bars) (p < 0.02). Data are mean from three or four patches.

FIGURE 3.

Modification by external MTSES and Au(CN)₂⁻. Sample whole cell currents were recorded at +30 mV for Cys-less (A), T338C (B), and L102C (C). CFTR currents were activated by application of cAMP stimulatory mixture (indicated as cAMP) in each case. A, Cys-less CFTR currents were insensitive to high concentrations of MTSES (2 mm) and Au(CN)₂⁻ (1 mm) but were confirmed as being carried by CFTR by their sensitivity to GlyH-101 (50 μm). B, T338C currents were inhibited by low concentrations of MTSES (1 μm) or Au(CN)₂⁻ (200 nm). C, L102C currents were insensitive to MTSES (2 mm) but were inhibited by GlyH-101 (50 μm) and Au(CN)₂⁻ (10 μm). All substances were added directly to the extracellular side of the membrane.

FIGURE 4.

Rate of modification of T338C by external MTSES. A, time course of whole cell current amplitude decay (measured at +30 mV, see Fig. 3) following application of 1 μm MTSES in different channel constructs. Current amplitudes have been scaled to amplitude prior to MTSES application (I_REL). The decline in current amplitude following MTSES application has been fitted by a single exponential function (red line). B, average modification rate constants (k) for MTSES, calculated from fits to data such as those shown in A. Modification rate constant for T338C/E1371Q was quantified from experiments using a higher concentration of MTSES (200 μm). Asterisks indicate a significant difference from T338C alone (p < 0.0005). Data are mean from three or four patches.

FIGURE 5.

Rate of modification of T338C and L102C by external Au(CN)₂⁻. A and B, time course of whole cell current amplitude decay (measured at 30 mV, see Fig. 3) following application of Au(CN)₂⁻ (200 nm in A, 10 μm in B) in different channel constructs. Current amplitudes have been scaled to amplitude prior to Au(CN)₂⁻ application (I_REL). The decline in current amplitude following Au(CN)₂⁻ application has been fitted by a single exponential function (red line). C, average modification rate constants (k) for Au(CN)₂⁻, calculated from fits to data such as those shown in A and B. Modification rate constants for T338C/E1371Q and L102C/E1371Q were quantified from experiments using a higher concentration of Au(CN)₂⁻ (100 μm). Asterisks indicate a significant difference from T338C and L102C alone as applicable (black bars) (p < 0.01).Data are mean from three or four patches.

FIGURE 6.

Relationship between modification rates and NBD function. Each panel illustrates the change in modification rate constant for the same reporter cysteine (T338C in A and C, L102C in B and D) in three different backgrounds (K464A, Cys-less, and E1371Q), for modification by MTSES (A and B) or Au(CN)₂⁻ (C and D) applied to the intracellular (●, inside) or extracellular (○, outside) side of the membrane. Modification rate constants under different conditions are as shown in Figs. 1C (internal MTSES), 2C (internal Au(CN)₂⁻), 4B (external MTSES), and 5C (external Au(CN)₂⁻). Note that L102C was not apparently modified by extracellular MTSES (B; see Fig. 3C).

FIGURE 7.

Models of CFTR pore structure during gating. The implications of the current experimental findings, that T338C and L102C in the CFTR pore show increased access to the extracellular solution in the closed state and increased access to the intracellular solution in the open state, can be interpreted according to a number of different simple diagram models of channel function. A, in the simplest interpretation of the inward facing/outward facing configurations of the TMDs, the closed state is designated as outward facing, and the open state as inward facing, with physical exposure of Leu-102 (orange) and Thr-338 (blue) alternating between the extracellular (closed) and intracellular (open) side of the membrane. B, as an ABC protein, the channel can be envisioned as having two gates, one of which controls channel opening (the activation gate, red) and a vestigial gate (green) that closes when the activation gate opens. The gates are positioned to allow opening of the channel to be associated with an increase in accessibility from the cytoplasmic side of the membrane and a decrease of accessibility from the extracellular side of the membrane for residues located between the two gates. Possible physical locations of these two putative gates are discussed under “Discussion.” C, this slightly less schematic view incorporates key aspects of the first two models. Opening of the channel is suggested to be associated with a physical dilation of the intracellular part of the narrow pore region and a physical constriction of the outer mouth of the pore, causing Leu-102 and Thr-338 to move from a position that is exposed to the extracellular solution in closed channels to a physically constricted, more cytoplasmically exposed position in open channels.