The Behavior and Structure of the Band 3 Anion Transport Site: a ³⁵Cl and ³⁷Cl NMR Study

Year: 1985 DOI: 10.7907/j15r-rw02
The transport of ions across cellular and organellar membranes is a widespread and fundamental process in biology. The goal of the present work is a molecular picture describing the ion translocation event in band 3, the most heavily used ion transport protein in typical vertebrate systems. The strategy employed involves ³⁵Cl NMR, which is shown both theoretically and experimentally to be a sensitive probe of two microscopic events: 1) the migration of Cl⁻ from solution to the vicinity of a macromolecular binding site, and 2) the binding of Cl⁻ to the site. The technique reveals ³⁵Cl⁻ linebroadening due to two classes of Cl⁻ binding sites on isolated, native red cell membranes. One class is composed solely of low affinity Cl⁻ binding sites of unknown function (KD > > 0.5 M), while the other class is composed solely of band 3 transport sites (KD = 80 ± 20 mM) which are identified by their affinity for substrate (Cl⁻), competing substrates (HCO⁻₃, F⁻, Br⁻, I⁻) and inhibitor (4,4'-dinitrostilbene-2,2'-disulfonate, or DNDS). A ³⁵Cl NMR method is developed to ascertain the sidedness of Cl⁻ binding sites relative to a compartment barrier such as a membrane: this approach shows that the low-affinity and transport sites are each found on both surfaces of the membrane.

The sequence of events in the Cl⁻ transport cycle is investigated by monitoring the behavior of the transport sites when the concentration of DNDS, p-nitrobenzenesulfonate (pNBS), Cl⁻, Br⁻, or H⁺ is varied. DNDS and pNBS, which are known to bind to outward-facing transport sites, each recruit all of the transport sites on both sides of the membrane to the inhibited outward-facing conformation, indicating that the inward- and outward-facing transport sites observed in the absence of inhibitor are merely different conformations of a single site. In addition, the transport sites on both sides of the membrane together behave like a homogeneous population of sites when [Cl⁻], [Br⁻], or pH is varied. These results are quantitatively consistent with the pingpong model for the transport cycle (Gunn and Frolich (1979) J. Gen. Physiol. 74, 351-374), in which a single transport site alternates between the inward- and outward-facing states and can only change states when occupied by bound anion. The rates of Cl⁻ binding and dissociation at both inward- and outward-facing transport sites are investigated with ³⁵Cl and ³⁷Cl NMR, and it is shown that each of these rates exceeds 10⁵ events sec⁻¹ site⁻¹ -- much faster than the known turnover rate of the chloride transport cycle (430 events sec⁻¹ site⁻¹, 0°C). Assuming that the rates of the influx and efflux half-turnovers of the transport cycle differ by 10² or less, it follows that the translocation of the chloride*transport site complex is the rate-limiting step in both half-turnovers (see Figure).

The structure of the transport machinery is investigated using transport inhibitors and proteases. The reversible inhibitor niflumic acid (NIF) has no effect on the transport site linebroadening: this inhibitor slows the translocation of bound Cl⁻ in both the influx and efflux half-turnovers. The covalent, arginine-specific reagents phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) each eliminate the transport site linebroadening: PG modifies an essential residue in the transport site and CHD slows the migration of Cl⁻ between the site and solution. The observed PG-sensitivity and pH-dependence of the transport site linebroadening (pKA = 11.1 ± 0.1, [Cl⁻] = 250 mM) indicate that an arginine residue provides the positive charge in at least one conformation of the transport site. A search is conducted for the minimal structure containing the intact transport site: this search begins with the removal of an innessential part of the transport domain, followed by monitoring of the transport site linebroadening for change. A variety of treatments leave some or all of the transport site linebroadening intact, including: 1) removal of the red cell membrane nonintegral proteins, 2) proteolytic removal of the soluble N-terminal domain of band 3, or 3) extensive proteolysis of band 3 by papain, which reduces band 3 to its transmembrane peptides (3-9 kDa). These results indicate that the essential arginine, as well as all other residues essential for Cl⁻ migration and binding to the transport site, are located on the papain-generated transmembrane peptides. The structural data presented here strongly support a picture in which the transport site, including the essential arginine, is buried in the membrane where it is resistant to proteolysis; and access of the buried site to solution Cl⁻ is provided by a channel that can be blocked by CHD. In summary, the minimal sequence of events in the Cl⁻ transport cycle can be schematically illustrated by

[Illustration. See abstract in scanned thesis for details]

A model is also presented that describes the molecular details of the ion translocation event: the translocation is proposed to begin when the transport site positive charge is neutralized by anion binding, so that a sliding hydrophobic barrier can move past the site and thereby expose the site to the opposite solution, as illustrated by

[Illustration. See abstract in scanned thesis for details]

A sliding barrier model could explain the translocation event in many other membrane transport systems as well.