High-chloride concentrations abolish the binding of adenine nucleotides in the mitochondrial ADP/ATP carrier family

Abstract

The ADP/ATP carrier (AAC) is a very effective membrane protein that mediates the exchange of ADP and ATP across the mitochondrial membrane. In vivo transport measurements on the AAC overexpressed in Escherichia coli demonstrate that this process can be severely inhibited by high-chloride concentrations. Molecular-dynamics simulations reveal a strong modification of the topology of the local electric field related to the number of chloride ions inside the cavity. Halide ions are shown to shield the positive charges lining the internal cavity of the carrier by accurate targeting of key basic residues. These specific amino acids are highly conserved as highlighted by the analysis of multiple AAC sequences. These results strongly suggest that the chloride concentration acts as an electrostatic lock for the mitochondrial AAC family, thereby preventing adenine nucleotides from reaching their dedicated binding sites.

Figure 1

Inhibitory effect of NaCl on the [α-³²P]ATP uptake by AtAAC1. (Inset) Time dependence of [α-³²P]ATP uptake into intact IPTG-induced E. coli cells harboring plasmid encoding mature AtAAC1 (squares) or the control plasmid (circles). The data shown is the mean of four independent experiments (SE < 10% of the mean values; see Supporting Material).

Figure 2

The electric properties of apo-bAAC1 differ as a function of the salt concentration. (A) Orientation of the bAAC1. A molecular-surface rendering and a ribbon representation are used for regions of the protein located, respectively, behind and in front of the plane in which the electric field is shown. The basic patches are highlighted in cyan. The cross-sectional view of the electric field after 30 ns differs between (B) 0.1 M and (C) 0.6 M assay. Maps of the electric field were obtained using the PMEPot (18) module of VMD (19) and OpenDX (http://www.opendx.org).

Figure 3

The series of ligand-association assays at 0.6 M NaCl results in the binding of ADP³⁻ only after removal of the ions. (A) The different initial positions of ADP³⁻ are depicted as colored tubes. A molecular-surface rendering is used for the protein (white), the upper (light-blue), and the lower patches (ice-blue). Chloride ions are shown as green van der Waals spheres. (B) Association experiment C. The starting position of ADP³⁻ and its final positions at 0.6 M NaCl and without salt are shown, respectively, as van der Waals spheres, and green and orange tubes. Image rendering using VMD (19).