Formation of a Chloride-conducting State in the Maltose ATP-binding Cassette (ABC) Transporter

Abstract

ATP-binding cassette transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ATP-binding cassette transporter into a channel.

Keywords: ABC transporter; ATPase; alternate access; ion channel; lipid; membrane.

FIGURE 1.

Conformation of MalF500. a, the disulfide bond between MalF_V442C and MalG_V230C (stars) indicates that the transporter is inward-facing. The disulfide bond between MalF_A394C and MalG_T182C indicates that the transporter is outward-facing. b, the MalFGK₂ complex in proteoliposome (4.5 μm) was treated with CuPhe₃ (100 μm) with or without ATP (2 mm), followed by addition of N-ethylmaleimide (5 mm) at the indicated time. The MalF-MalG cross-link products were detected by SDS-PAGE (15%) and Coomassie Blue staining. The cross-link intensity was quantified using ImageJ. The cross-link efficiency of MalFGK₂ (V230C-V442C) without ATP is taken as reference point (100%) in the top panel. The cross-link efficiency of MalF500 (T182C-A394C) with ATP is taken as reference point (100%) in the bottom panel. Each cross-link experiment was repeated at least three times.

FIGURE 2.

Activity of MalF500. a, effect of MalF500 on cell growth. E. coli BL21 was transformed with pBAD22-derived plasmids containing the MalFGK₂ complex under control of the arabinose promoter. Cells were grown in LB medium to OD_{600 nm} ∼1.0, serial-diluted, and plated on an LB-agar plate in the presence of 0.02% arabinose to induce expression of the MalFGK₂ complex. Plates were incubated at 37 °C for 16 h. b, ATPase activity. Proteoliposomes (0.5 μm) and maltose (1 mm) were incubated at 37 °C in the presence or absence of MalE (1 μm) as indicated. Standard deviation was obtained from three separate experiments. ND, not detected.

FIGURE 3.

Permeability of MalF500 in spheroplast assays. a and b, E. coli KM9 (unc-) overproducing the indicated complex was converted to spheroplasts and then diluted into an iso-osmotic solution of KCl (293 mm) in the absence (a) or presence (b) of valinomycin (15 μm). Cell lysis was monitored every 5 s at 540 nm. The rate of lysis is determined from the linear part of the curve (i.e. first 30 s after dilution in KCl).

FIGURE 4.

Ion channel activity of MalF500 in planar lipid bilayers. a, the electrical currents were recorded across a planar lipid bilayer (70% DOPC, 30% DOPG) at a holding membrane potential of +50 mV. Current traces were filtered at 500 Hz. The traces for the wild-type MalFGK₂ were recorded after >15 fusion events using nystatin/ergosterol as a reporter system indicating protein delivery. b, histogram of current amplitudes. The number of channel events obtained at +50 mV was determined using the Clampfit analysis program and the single channel search function. The currents were plotted as a function of their intensity. c, current-voltage curve for MalF500. Current amplitudes (I (pA)) were plotted according to the applied holding voltage. The slope of the curve represents the channel conductance in picosiemes. The reversal potential is −38 mV, as indicated by the x intercept of the curve.

FIGURE 5.

The periplasmic gate seals MalF500. a, the three gating residues (red, MalF_V442, MalG_T228, and MalG_V230) forming the periplasmic gate on MalF (yellow) and MalG (pink) were mutated to glycine residues. The space-filling representation was created with PyMOL using the inward-facing crystal structure of MalFGK₂ (PDB code 3FH6). b, growth curve of E. coli overproducing the indicated complex. Cells were grown in LB liquid medium at 37 °C for 150 min before induction with 0.2% arabinose. c, ATPase activity. Proteoliposomes containing the indicated mutant complex (0.5 μm) were incubated with maltose (1 mm) at 37 °C with or without MalE (1 μm) as indicated. Standard deviation was obtained from three separate experiments. d, spheroplast assays. Spheroplasts were diluted into an iso-osmotic solution of KCl in the presence or absence of valinomycin (15 μm). Cell lysis was monitored every 5 s at 540 nm.

FIGURE 6.

Ion channel activity of MalF500_GGG. a, typical electrical currents. Traces were obtained at a holding potential of −50 mV. b, all point histogram of current amplitudes. The number of channels present in the MalF500_GGG bilayer (≈25 channels incorporated, −250 pA) results in a higher total current amplitude than observed for MalF500 (≈3 channels incorporated, −15 pA). For MalF500_GGG, the observed channels are predominantly open, therefore concurrent closing events are rare, and the all-point histogram shows only two current distributions despite the presence of multiple channels. The number of channels incorporated is determined by dividing the maximum observed current by the amplitude of one opening event, as observed in the traces represented in a.