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.

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.

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.

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

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.

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.)
Questions:
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.

Questions:
(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    

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).

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., F₁F_o-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.)

• 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 P_i 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.

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⁺⁺.

SERCA

The primary structure of gramicidin A:
HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-NHCH₂CH₂OH
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.

Questions:
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.

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.

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).

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. 

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

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