Transcript Active transport

Chapter 11
• Membrane Transport of Small Molecules and the
Electrical Properties of Membranes
I.
Principles of membrane transport
- protein-free lipid bilayers are highly impermeable to ions
- two main classes of membrane transport proteins
- transporters ( also called carriers or permeases) and channels
- active transport – mediated by transporters coupled to an energy source
- passive transport – mediated by all channels and many transporters
II.
Transporters and passive transport
- glucose transporters
III.
Transporters and active transport
- coupled transporters; ATP-driven pumps; light-driven pumps
- uniporters; symporters; antiporters
- Na+-glucose cotransporter – coupled transport; antiporter
- lactose permease - coupled transport; symporter
- three classes of ATP-driven pumps
- Ca2+ pump
- Na+-K+ pump
- ABC transporters
IV.
Ion channels
- ion channels are selective and gated
- membrane potential
- K+- channel
- aquaporins
- neuron
- voltage-gated Na+-channels
- transmitter-gated ion channels
- neuromuscular transmission
Principles of membrane transport
Ion concentration differences across the lipid bilayer are useful for:
1. Driving various transport processes
2. Conveying electrical signals in electrically excitable cells
3. Making most of the cell’s ATP
4. Cell signaling
The rate at which a molecule diffuses across a synthetic lipid bilayer depend on its
size and solubility
The smaller the molecule and the less polar it is, the more rapidly it diffuses across the
bilayer
Permeability coefficients (in cm/sec)
through synthetic lipid bilayers
Product of the concentration difference
(in mol/cm3) and permeability coefficient
(in cm/sec) gives the flow of solute in
moles per second per square centimeter of
membrane
Two main classes of membrane transport proteins: Transporters and Channels
All these proteins are multi-pass transmembrane proteins
1. Transporters bind to a specific solute and undergo a series of conformational changes.
2. Channel proteins interact with the solute much more weakly; form aqueous pores; transport
at a much faster rate.
Solutes cross membranes by passive or active transport
An electrochemical gradient combines the membrane potential
and the concentration gradient
Transporters and passive transport
A conformational change in a transporter could mediate
the passive transport of a solute
The kinetics of simple diffusion and transporter-mediated diffusion
Transporters and active transport
Cells carry out active transport in three main ways:
(1)
Coupled transporters couple the uphill transport of one solute
across the membrane to the downhill transport of another.
(2)
ATP-driven pumps couple uphill transport to the hydrolysis of ATP.
(3)
Light-driven pumps, found mainly in bacteria and archaea,
couple uphill transport to an input of energy from light.
Cells drive active transport in three ways
The actively transported molecule is shown in yellow and the
energy source is shown in red
- ion-driven transporters mediate secondary active transport
- ATP-driven transporters mediate primary active transport
Three types of transporter-mediated transport
Uniports, symports, and antiports are used for both passive
and active transport
Active transport
Coupled transport – the transport of one solute strictly depends
on the transport of a second. It involves either the simultaneous
transfer of a second solute in the same direction (symporters) or
the transfer of a second solute in the opposite direction (antiporters)
The tight coupling between the transport of the two solutes allows
these carriers to harvest the energy stored in the electrochemical
gradient of one solute, typically an ion, to transport the other
The glucose-Na+ symport protein uses the electrochemical Na+
gradient to drive the import of glucose
The active transport of many sugars and amino acids into bacterial
cells is driven by the electrochemical H+ gradient across the
plasma membrane
E. coli lactose permease
During the transport cycle, some of the helices undergo sliding motions causing them
to tilt. An alternative opening and closing of the crevice between the helices exposes
the binding sites for lactose and H+, first on one side of the membrane and then the other
An asymmetric distribution of carrier proteins underlies the
transcellular transport of solutes
Three classes of ATP-driven pumps
The Ca2+ pump
Structures of the unphosphorylated, Ca2+-bound state (left) and the phosphorylated,
Ca2+-free state (right) have been determined by x-ray crystallography
Model showing how ATP binding and hydrolysis cause drastic conformational changes,
bringing the nucleotide-binding and phosphorylation domains into close proximity. This
change is thought to cause a 90° rotation of the activator domain, which leads to a
rearrangement of the transmembrane helices. The rearrangement of the helices disrupts
the Ca2+-binding cavity and releases the Ca2+ into the lumen of the SR.
Structure and function in a Ca2+ pump
The Na+-K+ pump is a P-type transport ATPase
A model of the pumping cycle of the Na+-K+ pump
The Na+ - K+ pump is required to maintain osmotic balance
and stabilize cell volume
ABC transporters
A section of the double membrane of E. coli
Auxiliary transport system associated with transport ATPases in bacteria
with double membranes
The transport ATPases belong to the ABC transporter supefamily
Examples of a few ABC proteins
Nature Structural & Molecular Biology 11, 918 - 926 (2004)
A typical ABC transporter consists of four domains – two highly hydrophobic
domains and two ATP-binding catalytic domains
ATP binding leads to dimerization of the two ATP-binding domains and ATP
hydrolysis leads to their dissociation.
Structure of an ABC transporter
In this transporter, the two TMDs (BtuC)
are identical to each other, as are the two
NBDs (BtuD), giving a clear two-fold
symmetry. The TMD dimer is gold and
yellow and the NBD dimer is magenta
and blue. Each TMD is composed of ten
membrane-spanning a-helices (most
ABC transporters have fewer than ten ahelices per domain) and together they
form an aqueous chamber in the
membrane that is open to the
extracellular surface. The NBDs form a
dimer with the two ATP-binding pockets
sandwiched at the dimer interface. Two
molecules of cyclic tetravanadate (shown
in ball-and-stick and colored elementally)
mimic nucleotide bound in the two
pockets formed at the protein-protein
interface of the NBD dimer.
Schematic representation of the vitamin B12 transporter BtuCD of E. coli
Nature Structural & Molecular Biology 11, 918 - 926 (2004)
Structure of the NBDs of ABC transporters
(a) The NBD closed dimer viewed from above, as if looking down through the membrane and TMDs
(which are hidden for clarity). As no complete transporter with bound ATP has been crystallized, a
consensus structure based on structures of several NBDs and modeled for P-gp17 is shown. The two ATP
molecules and two magnesium ions, shown in ball-and-stick and colored by atom types, occupy the two
nucleotide-binding pockets at the interface between the two NBDs and are coordinated by residues from
both NBDs (see also b). (b) NBD1 (blue) viewed from NBD2 (which is hidden for clarity). The
intracellular loops of TMD1 that form an interface with NBD1 are yellow (toward top). The residues and
motifs coordinating the Mg-ATP are shown as stick models and colored as follows: stacking aromatic
(Tyr401, light purple), Walker A motif (427-GSGCGKST-435, green), Walker B motif (551-ILLLDEAT558, yellow), Q-loop glutamine (Gln475, dark purple), H-loop histidine (His587, dark blue). The ABC
signature motif of this NBD (531-LSGGQ-535, orange) is not involved in coordinating the Mg-ATP in
b, but does contact the Mg-ATP (not shown) bound to the second nucleotide-binding pocket.
Nature Structural & Molecular Biology 11, 918 - 926 (2004)
The ATP switch model for the transport cycle of an ABC transporter
The schematic is for a drug exporter. The drug-binding site (red) is high-affinity and faces the inner leaflet of the
membrane. Step I: the transport cycle is initiated by binding of substrate to its high-affinity site on the TMDs from the
inner leaflet of the membrane. The affinity of the NBDs for ATP is increased, effectively lowering the activation energy
for closed dimer formation. Two molecules of ATP bind, cooperatively, to generate the closed dimer. Step II: the closed
NBD dimer induces a conformational change in the TMDs such that the drug-binding site is exposed extracellularly and
its affinity is reduced, releasing the bound drug. Step III: ATP is hydrolyzed to form a transition-state intermediate.
Hydrolysis of the two ATP molecules is normally sequential, although for some ABC transporters only one ATP may be
hydrolyzed. Step IV: sequential release of Pi, and then ADP, restores the transporter to its basal configuration.
Nature Structural & Molecular Biology 11, 918 - 926 (2004)
Ion channels
Can transport up to 100 million ions per second, a rate 105 times
greater than that mediated by a carrier protein
Among their many functions, ion channels control the
pace of the heart, regulate the secretion of hormones into
the bloodstream, and generate the electrical impulses
underlying information transfer in the nervous system.
Ion channels are ion-selective (ion selectivity) and fluctuate
between open and closed states (gated)
Ion channels, like enzymes, have their specific substrates:
potassium, sodium, calcium, and chloride channels permit only
their namesake ions to diffuse through their pores. The ability of
channels to discriminate among ions is called ion selectivity.
The atomic radius of K+ is 1.33 Å and that of Na+ is 0.95 Å
With only this difference to work with. K+ channels manage
to select for the K+ ion over the Na+ ion by a factor of more
than 1000.
Ion Channel Gating
Ion channel gating refers to opening and closing of the ion
conduction pore in response to a specific stimulus.
Certain channels open when ligands bind (ligand-gated
channels); others open in response to membrane voltage
(voltage-gated channels); a few open in response to mechanical
stress (mechanically gated channels)
Converting sound and movement into electrical signals
a, The hair cell has an array of pencil-shaped stereocilia on its surface, each linked to its neighbour through a 'tip
link'. b, The ion channel that mediates the conversion of sound or movement into electrical signals is located at
one (and possibly both) ends of the tip link, which is shown here as a relatively stiff connection. The channel pore
through which calcium (Ca2+) and potassium (K+) ions are transported is probably an assembly of four proteins,
with TRPA1 as at least one of the subunits. The mechanism by which this channel is controlled is speculative, but
it may involve a spring-like structural feature (with ankyrins forming the spring) and several other key proteins
coupled into the actin core of the stereocilium.
Membrane potential – difference in electrical charge on the two
sides of a membrane as a result of active electrogenic pumping
and passive ion diffusion
The equilibrium condition, in which there is no net flow of ions
across the plasma membrane defines the
resting membrane potential
The Nernst equation expresses the equilibrium condition
quantitatively
The number of ions that move across the plasma membrane
to set up the membrane potential is minute and the
movements of charge are generally rapid
Potassium channels are tetramers of identical subunits
in which specific "signature sequence" amino acids are
responsible for potassium selectivity
The signature sequence is conserved in all potassium channels
throughout nature and forms a structural unit
called the selectivity filter
The three-dimensional structure of a bacterial K+ channel shows
how an ion channel can work
K+ specificity of the selectivity filter in a K+ channel
In the vestibule, the ions are hydrated. In the selectivity filter, the carbonyl oxygens are
placed precisely to accommodate a dehydrated K+ ion. The dehydration of the K+ ion
requires energy, which is precisely balanced by the energy regained by the interaction
of the ion with the carbonyl oxygens that serve as surrogate water molecules
EACH OF THE binding sites closely mimics potassium ions'
octahedral hydration shell, thereby minimizing the energy required
to strip off their water coats.
Because of their smaller size, sodium ions don't fit in these binding
sites as snugly and thus find the energetic cost of trading their water
coat for a spot in the selectivity filter too high.
From the x-ray structure of the KcsA potassium channel at
2.0-Å resolution it was determined that the selectivity filter
is a narrow, 12-Å-long segment of the pore that is lined with
carbonyl oxygen atoms.
These atoms act as surrogate water molecules, allowing
potassium ions to shed their hydration shell and enter into
the pore.
Two potassium ions bind in the selectivity filter at once,
usually with an intervening water molecule. Entry of a
potassium ion from one side of the filter is associated with
potassium ion exit from the opposite side, resulting in rapid,
efficient potassium ion conduction.
Electrostatic repulsion between ions apparently prevents the
potassium ions from binding too tightly, allowing high
throughput in the setting of high selectivity.
A marvelous electrostatic feature of the potassium channel
is a cavity of water half way across the membrane with
specifically oriented a helices.
This design reduces the dielectric barrier, that is, the
energetic cost of moving an ion from the high-dielectric
environment of water to the low-dielectric membrane, and
thereby supports a high ion throughput.
Ion selectivity
K+ channels specifically conduct K+ ions because the
selectivity filter contains multiple binding sites that mimic a
hydrated K+ ion’s hydration shell.
Potassium channels achieve high conduction rates by exploiting
electrostatic repulsion between closely spaced ions and by
coupling the conformation of the selectivity filter to ion binding
within the filter
Gating
Large conformational changes within the membrane underlie
pore opening in K+ channels. Inner helices obstruct the pore
in the closed state and expand its intracellular diameter in the
opened state.
Different gating domains – ligand binding and voltage sensingappear to bring about a similar conformational change in the
pore of K+ channels
Voltage sensing
Conformational changes underlying voltage sensing in KvAP
are large and involve movements of arginine residues through
the membrane, near the protein lipid interface
Conventional and novel view on opening a voltage-gated K+ channel(A) K+ channel subunit, showing the six
transmembrane segments (S1–S6). Four (S1–S4) form voltage-sensor module with S4 as gating-charge carrier. S5 and
S6 form rigid-pore module at centre of channel.(B) Conventional (upper panel) and MacKinnon view (lower panel) on
opening. Pore region includes selectivity filter close to external surface, wide internal vestibule, and gate at internal
surface. Conventional view assumes that S4 is located in oily interior of the protein and moves outward at positive
internal potentials (helical screw model), causing gate to open. MacKinnon view assumes that S4 is connected with S3
and forms paddle that moves upward in lipid environment at positive potentials.
Comparison of Cl- and K+ channel architectures
Aquaporins are permeable to water but impermeable to ions
Aquaporins play a key role in cellular water homeostasis in humans,
animals, and plants. These water channel proteins associate as tetramers.
Each aquaporin contains a single 28-Å-long cylindrical pore that supports a
string of nine hydrogen-bonded water molecules in single file.
The structure of aquaporins
Animations of the movement of water through aquaporins:
The protein backbone of one subunit of bovine aquaporin 1 is shown as a ribbon,
with the dimensions of the water pore shown in blue dots. At right, water molecules
(space-filling models) move through the pore single file. Residues defining the
constriction region are shown as ball-and-stick representations. In particular, a
histidine protruding into the pore ensures that molecules larger than water can't
enter. (Gray = carbon, blue = nitrogen, red = oxygen.)
High-resolution structures reveal the inner workings of aquaporins in
atomic detail
Water molecules travel single file through a narrow pore that connects
water-filled intracellular and extracellular vestibules. This 28-Å-long pore
is lined with a row of eight carbonyl oxygen atoms that can accept
hydrogen bonds from the queue of water molecules, "ensuring that every
water molecule is precisely oriented throughout the channel." But because
the rest of the pore is lined with mostly hydrophobic amino acids, "waterpore interactions are kept to a minimum," allowing water to rush through
each pore at a rate of 3 x 109 molecules per second.
FOR A SHORT STRETCH, the pore narrows to just wide enough for
a water molecule to pass, preventing larger molecules from entering.
And ions--which would have to abandon their bulky water coats to
enter--don't do so because the pore isn't a good substitute. In addition,
a conserved, positively charged arginine residue in this region prevents
cations, including protonated water (H3O+), from entering.
Nature has gone to great effort to prevent protons from passing through,
too. Previous structural work had suggested that a pair of asparagine
residues as well as the dipole moments of short a-helices would force water
molecules to flip near the pore's center. This forced reorientation breaks
the continuous chain of hydrogen-bonded waters and prevents protons
from "hopping" along the hydrogen-bond network.
PROTON BLOCK
Simulation of water molecules passing through an aquaporin suggested that
the central water molecule is forced out of line in part by hydrogen bonds
donated by a pair of asparagines and this defect in the hydrogen-bonded
water wire caused the exclusion of protons in these channels.
Recent simulations of protons passing through the protein have called this
mechanism into question.
Normally, continuous, single-file columns of water like these conduct
protons.
Protons can easily hop along a similar single-file water column in the water
pore of gramicidin A. But unlike gramicidin A, an antibiotic peptide that
kills bacteria by forming pores in their cell membranes, aquaporins must
not leak protons. Doing so would disturb the delicate balance of charge
across cell membranes that underlies cellular function.
So how do aquaporins manage to do what gramicidin A can't?
The water column in aquaporins does have some distinguishing features.
Near the center of the aquaporin pore, hydrogen bonds from a pair of
asparagine residues (as well as the pull of nearby a-helix dipoles)
reorient the central water molecule, preventing it from accepting a
proton from nearby water molecules.
From structural observations, it was suggested that the forced
reorientation of this central water molecule might break the continuous
chain of hydrogen-bonded waters and possibly prevent protons from
"hopping" along the hydrogen-bonded water "wire."
The barrier to proton transfer is highest in the part of the aquaporin
pore that boasts the highest electrostatic potential: the region centered
around the two central asparagines and the partially positively charged
a-helix dipoles, known as the NPA region.
Recent results indicate that the main barrier to proton transfer is not
caused by interruption of the hydrogen-bonded water chain, as had
previously been speculated, but rather by an electrostatic field created
by the a-helix dipoles in the NPA region.
The barrier to proton transfer in aquaporins is estimated to be 6–7 kcal
per mole--about the same as estimates of the barrier to proton
translocation across lipid membranes
A typical vertebrate neuron
Voltage-gated cation channels generate action potentials in
electrically excitable cells
- neurons
- muscle cells
- endocrine cells
- egg cells
An action potential is triggered by a depolarization of the plasma membrane – that
is, by a shift in the membrane potential to a less negative value.
Propagation of an action potential along an axon without attenuation
Action potentials are the direct consequence of the properties of
voltage-gated cation channels
The “ball-and-chain” model of rapid inactivation of a voltage-gated K+ channel
Myelination increases the speed and efficiency of action potential propagation
in nerve cells
Fluorescence micrograph and diagram of individual myelinated axons teased
apart in a nerve. Three different proteins are detected – voltage-gated Na+
channels stained in green are concentrated in the axonal membrane at the
nodes of Ranvier; an extracellular protein called Caspr, stained in red, marks
the end of each myelin sheath; voltage-gated K+ channels, stained in blue,
localize to regions in the axon plasma membrane close to the nodes
Patch-Clamp recording indicates that individual gated channels open
in an all-or-nothing fashion
Patch-Clamp measurements for a single voltage-gated Na+channel
Transmitter-gated ion channels convert chemical signals into electrical signals
at chemical synapses
A chemical synapse
Chemical synapses can be excitatory or inhibitory
Excitatory neurotransmitters open cation channels, causing an influx of Na+ that depolarizes the
postsynaptic membrane toward the threshold potential for firing an action potential.
Inhibitory neurotransmitters open either Cl- channels or K+ channels, and this suppresses firing by
making it harder for excitatory influences to depolarize the postsynaptic membrane.
A neuromuscular junction
The acetylcholine receptors at the neuromuscular junction are
transmitter-gated cation channels
Three conformations of the acetylcholine receptor
A model for the structure of the acetylcholine receptor
Neuromuscular transmission involves the sequential activation of five
different sets of ion channels