Molecular Biochemistry I

Membrane Transport

Contents of this page:
Ion carriers & ionophores
P-class ion pumps & carrier protein structures
Ion channels
Patch clamping

Note: This discussion of membrane transport will focus on selected examples of transport catalysts for which structure/function relationships are relatively well understood.

Transporters are of two general classes: carriers and channels. These are exemplified by two ionophores (ion carriers produced by microorganisms):


Valinomycin is a carrier for potassium. It is a circular molecule made up of 3 repeats of the sequence shown at right.

Puckering of the ring, stabilized by H-bonds, allows valinomycin to closely surround a single unhydrated K+ ion. Six oxygen atoms of the ionophore interact with the bound K+, replacing oxygen atoms of waters of hydration. See diagrams below right and in Voet & Voet, p. 731.

Valinomycin is highly selective for K+ relative to Na+. The smaller Na+ ion cannot simultaneously interact with all six oxygen atoms within valinomycin. Thus it is energetically less favorable for Na+ to shed its waters of hydration to form a complex with valinomycin.

Whereas the interior of the valinomycin-K+ complex is polar, the surface of the complex is hydrophobic. This allows valinomycin to enter the lipid core of the bilayer, to solubilize K+ within this hydrophobic milieu.

Explore the crystal structure of valinomycin with Chime at the Virtual Museum of Minerals and Molecules.

Valinomycin is a passive carrier for K+. It can bind or release K+ when it encounters the membrane surface. Valinomycin can catalyze net K+ transport because it can translocate either in the complexed or uncomplexed state. The direction of net flux depends on the electrochemical K+ gradient.

Proteins that act as carriers are too large to move across the membrane. They are transmembrane proteins with fixed topology. An example is the GLUT1 glucose carrier, in plasma membranes of various cells, including erythrocytes. GLUT1 is a large integral protein, predicted via hydropathy plots to include 12 transmembrane a-helices.

Carrier proteins cycle between conformations in which a solute binding site is accessible on one side of the membrane or the other. There may be an intermediate conformation in which a bound substrate is inaccessible to either aqueous phase.

With carrier proteins there is never an open channel all the way through the membrane.

The transport rate mediated by carriers is faster than in the absence of a catalyst, but slower than with channels. A carrier transports only one or a few solute molecules per conformational cycle, whereas a single channel opening event may allow flux of many thousands of ions.

Carriers exhibit Michaelis-Menten kinetics. You may review Michaelis-Menton kinetics in the Biochemistry Simulations tutorial at right.

Note: Hold down the Control key while clicking on the above icon.

Classes of Carrier Proteins:

Uniport (facilitated diffusion) carriers mediate transport of a single solute. An example is the GLUT1 glucose carrier. See diagrams p. 734. The ionophore valinomycin is also a uniport carrier.

Symport (cotransport) carriers bind two dissimilar solutes (substrates) and transport them together across a membrane. Transport of the two solutes is obligatorily coupled. A gradient of one substrate, usually an ion, may drive uphill (against the gradient) transport of a co-substrate. It is sometimes referred to as secondary active transport.

Examples include

  • the glucose-Na+ symport found in plasma membranes of some epithelial cells

  • the bacterial lactose permease, a H+ symport carrier.

uniport.gif (3448 bytes)

Lactose permease catalyzes uptake of the disaccharide lactose into E. coli bacteria, along with H+, driven by a proton electrochemical gradient. It is the first carrier protein for which an atomic resolution structure has been determined.

Lactose permease has been crystallized with thiodigalactoside (TDG), an analog of lactose.

  • The substrate binding site is at the apex of an aqueous cavity between 2 domains, each consisting of 6 transmembrane a-helices. In the conformation observed in this crystal structure, the substrate analog is accessible only to what would be the cytosolic side of the intact membrane.
  • Residues identified as being essential for H+-binding are also near the middle of the membrane. 

As in simple models of carrier transport based on functional assays, the tilt of transmembrane a-helices is assumed to change, shifting access of lactose & H+ binding sites to the other side of the membrane during the transport cycle. (See diagram of carrier transport above.)

Explore below the structure of lactose permease. This crystal structure was solved by J. Abramson, I. Smirnova, V. Kasho, G. Verner, H. R. Kaback, & S. Iwata in 2003 (PDB 1PV7).

Recommended display options (Use menus via the right mouse button):

Display as cartoon with color chain.
There are 2 copies of the permease. (This is an artifact of crystallization. The functional unit is a monomer.)
1. What secondary structure(s) is(are) present?
2. How much of the protein is likely to be embedded within the lipid membrane? (Hint: The intramembrane portion of the protein mainly consists of transmembrane a-helices.)

Select hetero-ligand; display as ball & stick with color CPK.
Note the position of the substrate analog (also selectable as TDG). TDG has a sulfur atom replacing the bridging oxygen atom of the normal disaccharide substrate.
To zoom in, hold down the shift key and left-drag the image.
To move the image, hold down the control key and right-drag.

Now select protein and display as spacefill.
Separately select protein-hydrophobic and protein-polar, and select -change color, giving each a different color.

(1) Is the substrate binding site for an individual permease simultaneously accessible to both sides of the membrane?
(2) Are surfaces facing the lipid bilayer, and the substrate-binding cavity, lined mainly with hydrophobic or polar amino acids?

     C   O   N   S    

Antiport (exchange diffusion) carriers exchange one solute for another across a membrane. Usually antiporters exhibit "ping pong" kinetics. A substrate binds and is transported across the membrane. Then another substrate binds and is transported in the other direction. Only exchange is catalyzed, not net transport, because the carrier protein cannot undergo the conformational transition in the absence of bound substrate.

An example is the adenine nucleotide translocase (ADP/ATP exchanger), which catalyzes 1:1 exchange of ADP for ATP across the inner mitochondrial membrane. (See class on ATP synthesis, & diagram p. 750).

Active transport enzymes couple net solute movement across a membrane to hydrolysis of ATP. An active transport pump may be a uniporter or an antiporter.

ATP-dependent ion pumps are grouped into classes, based on transport mechanism as well as genetic and structural homology. Examples include the P-class pumps to be discussed here, as well as F-class (e.g., F1Fo-ATPase to be discussed later) and related V-class pumps.

ABC (ATP binding cassette) transporters, which catalyze transmembrane movements of various organic compounds including amphipathic lipids and drugs, will not be discussed here. (For information about ABC transporters see a recent review.)

acttrans.gif (2619 bytes)

P-class ion pumps are a gene family exhibiting sequence homology. They include:

The reaction mechanism for a P-class ion pump involves transient covalent modification of the enzyme. 
  • At one stage of the reaction cycle, phosphate is transferred from ATP to the carboxyl of a glutamate or aspartate side-chain, forming a "high energy" anhydride linkage (~P). 
  • At a later stage in the reaction cycle, the Pi is released by hydrolysis. 

While other P-class pumps have been extensively studied (see textbook p.739-742 and reference list), this discussion will focus on the endoplasmic reticulum Ca++ pump, whose structure is known at atomic resolution.

The ER Ca++ pump is sometimes called SERCA, or Sarco(Endo)plasmic Reticulum Ca++-ATPase. (Muscle endoplasmic reticulum is called sarcoplasmic reticulum.)

The SERCA reaction cycle is summarized at right and below. In the diagram at right, conformational changes altering accessibility of Ca++-binding sites to the cytosol or ER lumen are depicted as positional changes. Keep in mind that SERCA is a large protein that maintains its transmembrane orientation.

  1. Two Ca++ ions bind tightly to the enzyme from the cytosolic side of the membrane, stabilizing the conformation that allows ATP to react with an active site aspartate residue.
  2. Phosphorylation of the active site aspartate residue induces a conformational change that shifts accessibility of the 2 Ca++-binding sites from one side of the membrane to the other, and lowers the affinity of the binding sites for Ca++.
  3. Ca++ dissociates into the ER lumen. 
  4. Ca++ dissociation promotes hydrolysis of Pi from the enzyme Asp residue and a conformational change (recovery) that causes the Ca++ binding sites to be accessible again from the cytosol.  
The X-ray structure of muscle SERCA (Ca++-ATPase) at right shows 2 Ca++ ions (colored magenta) bound between transmembrane a-helices in the membrane domain. The active site Asp351, which is transiently phosphorylated during catalysis, is located  in a cytosolic domain, far from the Ca++ binding sites.

SERCA structure has been determined in the presence and absence of Ca++, with or without substrate or product analogs and inhibitors. Substantial differences in conformation have been interpreted as corresponding to different stages of the reaction cycle.

  • Large conformational changes in the cytosolic domain of SERCA are accompanied by deformation and changes in position and tilt of transmembrane a-helices.
  • The data indicate that when Ca++ dissociates water molecules enter Ca++ binding sites, while charge compensation is provided by protonation of Ca++-binding residues.

The simplified cartoon at right at represents the proposed variation in accessibility and affinity of Ca++-binding sites during the reaction cycle. Only 2 transmembrane a-helices are represented, and the cytosolic domain of SERCA is omitted.

More complex diagrams & animations have been created by several laboratories, based on available structural evidence. For example, see:

  • animation provided by the lab of D. H. MacLennan.
  • diagram by C. Toyoshima, in a website of the Society of General Physiologists (select Poster).
  • website of the Toyoshima Lab (select Resources for movies).

Explore at right the crystal structure of muscle Ca++-ATPase (SERCA) with bound Ca++.


Introduction to Ion Channels

Channels cycle between open and closed conformations. When open, a channel provides a continuous pathway through the bilayer, allowing flux of many ions.

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Gramicidin is an example of a channel. It is an unusual peptide, with alternating D and L amino acids.  In lipid bilayer membranes, gramicidin dimerizes and folds as a right handed b-helix. The dimer just spans the bilayer (See below and Fig. 20-11 p. 732).

The primary structure of gramicidin A:
Note that the amino acids all have hydrophobic side-chains (R groups), D & L amino acids alternate, and both ends of the peptide are modified (blocked).

  • The outer surface of the gramicidin dimer, which interacts with the core of the lipid bilayer, is hydrophobic.
  • Ions pass through the more polar lumen of the helix.

The 3D structure of gramicidin (solved via solid state NMR by J. Prilusky in 1996, Protein Databank file 1MAG) is displayed at right.

Right click the image to select "chain" from the color menu, and display as "sticks".  Note the 2 gramicidin molecules joined head to head (via H-bonding at their formyl-N-termini).
Drag the image to view the channel from the side, and down its axis.

View also with color CPK, and with spacefill display.


Where are amino acid side-chains located in relation to the helix? (Hint: Look for Trp ring structures.)
What is the significance of this location?
What atoms line the helix lumen?
How does the location of specific amino acids relative to the lipid bilayer, such as those with partly polar side-chains (e.g., tryptophan) and those with aliphatic side-chains, compare to the location of such residues in a typical transmembrane a-helix? Compare to the Chime exercise in the section on lipids & membranes.

     C   O   N   H   
Ion flow through individual gramicidin channels can be observed if a small number of gramicidin molecules is present in a lipid bilayer separating two compartments containing salt solution. 

With transmembrane voltage clamped at some value, current (ion flow through the membrane) fluctuates. Each fluctuation, attributed to opening or closing of one gramicidin channel, is the same magnitude. The current increment corresponds to the current flow through a single channel. At right is a drawing - not actual data.

Gating (opening & closing) of a gramicidin channel is thought to involve reversible dimerization. An open channel forms when two gramicidin molecules join end to end to span the membrane. This model is consistent with the finding that, at high concentrations of gramicidin, the overall transport rate depends on [gramidicin]2.

Cellular channels usually consist of large, protein complexes with multiple transmembrane a-helices. Their gating mechanisms must differ from that of gramicidin.

Control of channel gating is a form of allosteric regulation. Conformational changes associated with channel opening may be regulated by one of the following:

The technique of patch clamping is used to study ion channel activity.

A narrow bore micropipet containing a salt solution may be pushed up against a cell or vesicle (diagram A at right), and then pulled back, capturing a fragment of membrane across the pipet tip (B at right).

pclamp.gif (3331 bytes)

A voltage is imposed between an electrode inside the patch pipet and a reference electrode in contact with the surrounding salt solution. Current is carried by ions flowing through the membrane. 

pcequip.gif (4016 bytes)
If a membrane patch contains a single channel with 2 conformational states, the current will fluctuate between 2 levels as the channel opens and closes, as represented below.

The increment in current, as the channel cycles between open and closed state, reflects the rate of ion flux through a single channel.

View a video of an oscilloscope image during a patch clamp recording.

Then carry out a Studio Exercise involving analysis of patch clamp data.

Copyright 1998-2007 by Joyce J. Diwan. All rights reserved.

Additional material on Membrane Transport:
Readings, Test Questions, Studio Exercise, & Tutorial

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