A Caltech Library Service

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


Falke, Joseph John (1985) The Behavior and Structure of the Band 3 Anion Transport Site: A ³⁵Cl and ³⁷CL NMR Study. Dissertation (Ph.D.), California Institute of Technology. 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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Davidson, Norman R.
Thesis Committee:
  • Davidson, Norman R. (chair)
  • Chan, Sunney I.
  • Hopfield, John J.
  • Beauchamp, Jesse L.
Defense Date:28 September 1984
Funding AgencyGrant Number
Record Number:CaltechTHESIS:02112019-112435563
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11389
Deposited On:11 Feb 2019 22:53
Last Modified:16 Apr 2021 22:15

Thesis Files

PDF - Final Version
See Usage Policy.


Repository Staff Only: item control page