Contents of this page:
Ion carriers & ionophores
P-class ion pumps & carrier protein structures
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.|
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.
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.
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):
cartoon with color
display as ball & stick with color
Now select protein and display as
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.)
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.
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.
|The X-ray structure of muscle
SERCA (Ca++-ATPase) at right
shows 2 Ca++
(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.
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:
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.||
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:
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
View also with color CPK, and with spacefill display.
| 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).
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.
|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