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Chapter 13
Membrane Channels and Pumps
Permeability of membrane is
conferred by two classes of
membrane proteins : PUMPs and
CHANNELs.
-PUMP : use ATP energy or light
absorption. = active transport.
- CHANNEL : enable ions to flow
through membranes in a
thermodynamically downhill
direction. = passive transport, or
facilitated diffusion.
- Pump : two types of ATP-driven pump, 1) P-type ATPase and
2)ATP-binding cassette(ABC) transporter. Pump can establish
persistent gradients of ions across membrane.
- Channel : can allow these ions to flow rapidly across
membranes down gradient.
The expression of transporters largely defines the
metabolic activities of a given cell type
- Each cell type express a specific set of transporters.
- Transporters largely determine the ionic composition and the compounds
inside cells  cell’s characteristic reactions
- Ex> GLUT(glucose transporter) have 5 homologs.
In case of GLUT3 of high affinity for glucose, expressed only on
neurons and a few other cell types. When glucose is present at low
concentration, first call on glucose.
13.1 The transport of molecules across a membrane
may be active or passive
- Two factors determine whether a molecule will cross a
membrane 1) the permeability of the molecule in a lipid bilayer
and 2) the availability of an energy source.
Many molecules require protein transporters to cross
membranes
-Simple diffusion : molecule will pass through a membrane
down their concentration gradient. Ex> lipophilic molecule,
steroid hormones.
-Passive transport : Charged ions pass through specific
channels down their concentration gradient. Channels display
substrate specificity. Ex> Na+ K+…
-Active transport : Charged ion must be pumped against a
concentration gradient(low conc. → high conc.) Energy is
required.
Free energy stored in concentration gradients can be
quantified
-To abtain concentration gradient,
requires an input of free energy
because of a decrease in entropy.
-Uncharged molecule ;
ΔG = RTln(c2/c1) = 2.303RTlog(c2/c1)
-Charged molecule ;
ΔG = RTln(c2/c1) + ZFΔV
= 2.303RTlog(c2/c1) + ZFΔV
R=gas constant
T=temperature in kelvins
Z=electrical charge
F=Faraday constant
ΔV= potential in volts across the membrane
A transport process must be active when ΔG is
positive.
13.2 Two families of membrane proteins use ATP
hydrolysis to pump ions and molecules across
membranes
-The ion gradients are generated by a specific transport system, an
enzyme that is called the Na+-K+ pump or the Na+-K+ ATPase.
-The hydrolysis of ATP by the pump provides the energy needed for
the active transport of 3 Na+ out of the cell and 2 K+ into the cell,
generating gradient (-50mV membrane potential maintenance).
-1/3 ATP consumed in resting animal
-P type ATPase : ATPase form a key
phosphorylated intermediate.
Phosphoryl group is linked to the side chain of
a specific conserved aspartate residue in the
ATPase. (SERCA, gastric H+-K+ ATPase)
P-type ATPases couple phosphorylation and conformational
changes to pump calcium ions across membranes
- Pump protein can exist in two principal conformational
states, one with ion-binding sites open to one side of the
membrane and the other with ion-binding sites open to the
other side.
<Ca2+ ATPase = SERCA (example of P-type ATPase)>
-In sarcoplasmic reticulum of muscle cell.
-Constitutes 80% of the SR membrane protein.
-Important role in muscle contraction.
-Maintain Ca2+ concentration 0.1uM in cytoplasm while 1.5mM in SR
- Ca2+ bound SERCA.
-Single 110kda polypeptide.
-Transmembrane = 10 α helices.
-2 Ca2+ binding domains at TM.
-3 cytoplasmic domain.
N=ATP nucleotide binding
P=accept the phosphoryl group on a
aspartate residue
A=actuator, linking changes in N & P
domains to the transmembrane part
-SERCA is structurally dynamic.
-The SERCA without Ca2+ and with a
phosphorylaspartate analog in the P domain.
-The transmembrane part of the enzyme has
rearranged and the well-organized Ca2+
binding site are disrupted.
Ca2+ bound
Ca2+ not bound
ATP bind.
Rearragement of
cytoplasmic domains.
Phosphoryl group is
transferred from ATP
to Asp351.
ADP release.
Conformational
change in
membrane domain.
Ca2+ release.
Ca2+ bind.
Enzyme everts back to
the E1 conformation.
Phosphoaspartate is
hydrolyzed. Pi release.
Digitalis specifically inhibits the Na+-K+ pump by blocking
its dephosphorylation
-Na+-K+ pump a2b2 tetramer, a subunit
homologous to SERCA
-Certain steroid derived from plants are
potent inhibitors.
-Digitoxigenin : cardiotonic steroid. Inhibit
the dephosphorylation of the E2-P form of
the ATPase.
-Digitalis : mixture of cardiotonic steroid
from the dried leaf of the foxglove plant.
Digitalis increases the force of contraction of heart muscle.
Inhibition of Na+-K+ pump → higher level of Na+ inside the cell
→ slower extrusion of Ca2+ by the Na+-Ca2+ exchanger →
increase in the intracellular level of Ca2+ → enhance the
ability of cardiac muscle to contraction.
Congestive heart failure (울혈성심부전증) 치료약
P-type ATPase are evolutionarily conserved and play a
wide range of roles
Yeast : 16 proteins that belong to the P-type ATPase family
(ca2+, Na+, Cu2+ ).
※ fippase : 5 members of this family.
Transport of phospholipids with amino acid
head group (ex, phosphatidylserine)  membrane
asymmetry
Human : 70 proteins that belong to the P-type ATPase family.
Multidrug resistance highlights a family of membrane
pumps with ATP-binding cassette domains
-Multidrug resistance : the development of resistance to one drug also
makes the cell less sensitive to a range of other compound.
-Multidrug-resistance protein or P-glycoprotein : 170kda. ATPdependent pump.
- MDR pump : 4 domains.
2 membrane spanning + 2 ATP binding
domain(=ATP-binding cassettes, ABCs).
-Largest single family in the E.coli
genome. Why? In Human?
-Eukaryotes vs prokaryotes
-Lipid transporter
-Dimer of 62kDa chains.
-N-term is membrane spanning
-C-term is ATP-binding casette.
-Two ABCs are in contact, but
do not interact strongly in the
absence of ATP.
-P-loop NTPase family
ABC transporter Structure
from Vibrio
(GXXXXGK, 1st b strand and 1st
helix)
Free of both ATP and
substrate.
Substrate enters the central
cavity. And conformational
change in the ABCs.(increase
affinity for ATP)
ATP bind.
Hydrolysis of ATP and
release of ADP and Pi
reset the transporter.
Strong interaction between ABCs →
conformational change in membrane
spanning domain. Substrate out.
13.3 Lactose permease is an archetype of secondary
transporters that use one concentration gradient to power the
formation of another
* cotransporter
Couple the downhill flow
of one species to the
uphill flow of another in
the opposite direction
across the membrane.
Use the flow of one
species to drive the flow
of a different species in
the same direction
across the membrane.
Like channel, Transport
a specific species in
either direction
governed only by
concentrations of that
species on either side
of the membrane.
- Lactose permease of E. coli. Symporter.
- Uses the H+ gradient across the membrane generated by the
oxidation of fuel molecules to drive the uptake of lactose and other
sugars against a concentration gradient.
-Two helves, each of which contains 6 membrane spanning α helices.
-Sugar lies in a pocket in the center of the protein.
A proton from outside the
cell binds to a residue in
the permease(Glu269).
Permease binds lactose
from outside the cell.
Structure
evert.
Permease
Evert.
Permease release a proton
to the inside of the cell.
Permease release lactorse
to the inside of the cell.
13.4 Specific channels can rapidly transport ions
across membranes
※ Channel : Membrane protein .
: Fast ion transport rate.
: Highly sophisticated molecular machines.
: Respond to chemical and physical changes and
undergo precisely timed conformational changes.
Action potentials are mediated by transient changes in Na+ and
K + permeability
-Nerve impulse : electrical signal produced by the flow of ions
across the plasma membrane of a neuron.
- Resting state : High K+ concentration, low Na+ concentration
inside of the cell(membrane potential : -60mV).
- Depolarization : Na+ flow into the cell(Na+ channel open,
membrane potential : +30mV).
- Repolarization : K+ flow outward(K+ channel open, membrane
potential : -60mV)
Patch-clamp conductance measurements reveal the activities
of single channels
To confirm the existence of the channel,
※Patch clamp technique : measure the ion conductance
through a small patch of cell membrane.
(by Erwin Neher and Bert Sakmann in 1976)
Single ion channel undergoing transitions
between closed and open states.
Patch pipette
diameter : 1μm
The activity of the channels in
the entire plasma membrane can
be monitored.
A piece of plasma membrane with
its cytoplasmic side now facing
the medium is monitored.
-The flow of ions through a single channel can be monitored
with a time resolution of microseconds.
-First views of single biomolecule activities
The structure of a potassium ion channel is an archetype for
many ion-channel structures
※Na+ channel
- purified from the electric organ of electric eel.
- bind a specific neurotoxin
(= Tetrodotoxin from puffer, 10ng lethal dose).
- single 260kda chain.
- contains 4 internal repeats(similar a.a. seq. gene duplication)
- each repeat contains 5 hydrophobic segment(S1, S2,S3, S5, S6).
- each repeat contains 1 highly positive charged segment(S4).
- S1~S6 = membrane-spanning α helices.
- S4 = voltage sensors of the channel.
※ K+ channel
- 70 kDa protein that has 4 subunits(= tetramer).
- S1~S6 = membrane-spanning α helices.
- homologous to one of the repeated units of Na+ channels.
- S5 and S6 = the actual pore of the K+ channel.
- S1~S4 = apparatus that open the pore.
※ K+ channel (tetramer)
Roderick MacKinnon determined the structure of a
bacterial K+ channel in 1998  Nobel prize 2003
The structure of the potassium ion channel reveals the basis
of ion specificity
※ K+ channel
Outside of the cell
K+
K+ ions must give up their
water molecules and interact
directly with groups from the
protein.
Pore becomes more
constricted(3Å).
Inside of the cell
K+ ion can fit in the pore
without losing its shell of
bound water(10Å).
-Selectivity filter : determines the preference for K+ over
other ions (100-fold than Na+).
-Conserved seq. : TVGYG extended confromation,
carbonyl oxgens line up.
- K+ channels pass only K+.
- Na+ is smaller than K+.
- How is this high selectivity achieved?
-Answer : Free energy costs of dehydrating these ions
are considerable.
(Na+ =301kJ/mol, K+ =230kJ/mol)
- The channel pays the cost of dehydrating K+ by
providing compensating interactions with the carbonyl
oxygen atoms lining the selectivity filter.
The structure of the potassium ion channel explains its rapid
rate of transport
High selectivity needs tight binding site!
How to achieve fast transport of channels?
Hydrated K+ ion
proceeds into the
channel.
The ion then gives up its coordinated
water molecules and binds to a site
within the selectivity filter region
The ion can move between
the four sites(tetramer,
similar ion affinity).
This multiple binding site mechanism solves the apparent paradox of
high ion selectivity and rapid flow.
Voltage gating requires substantial conformational changes in
specific ion-channel domains
- S1~S4 forms domains termed “paddles”.
- S4 = voltage sensor, highly positive charged residues.
Model for voltage gating of ion channels
PADDLEs (S1-S4) include S4, the voltage sensor
※ Close state : paddles lie in
※ Depolarization : the paddles are
a “down” position.
pulled through the membrane into
an “up” position. This motion pulls
the base of the channel apart
openining the channel
A channel can be inactivated by occlusion of the pore :
The ball-and-chain model
-Na+ and K+ channels undergo
“inactivation” within miliseconds.
-Cleavage of cytoplasmic side by
trypsin → stayed persistently
activation(channel always open).
-Inactivation was restored by
adding a synthetic peptide
corresponding to the first 20 a.a
cytoplasmic peptide
Ball-and-stick model
- The first 20 residues of the K+ channel form a
cytoplasmic unit(the ball) that is attached to a flexible
segment of the polypeptide(the chain).
- Channel is closed →
the ball rotate freely.
- Channel open → the
ball quickly finds a
complementary site in
the open pore.
And occludes it.
Chain length & speed?
Ball is postive charge?
The acetylcholine receptor is an archetype for ligand-gated
ion channels
-Neurotransmitter : small, diffusible signal molecules between a
neuron and another neuron.
-Ex> Acetylcholine
-Synaptic cleft : a gap of about 50nm
between presynaptic and postsynaptic
membrane.
-Nerve impulse → export of the contents
of some 300 vesicles of acetylcholine into
the cleft.
-Acetylcholine receptor at postsynaptic
membrane.
※Acetylcholine receptor
-Ligand-gated channel.
-Purified from electric ray.
-268kDa, pentamer of 4 kinds of membranespanning subunit(α2, β,γ, and δ).
-Each subunit has a large extracellular
domain + 4 hydrophobic segments(membrane
spanning) + intracellular segment.
α2, β,γ, and δ
- Pentamer, five-fold symmetry.
-Binding of acetylcholine to the extracellular domain →
structural alteration(rotations of the membrane spanning
helices).
- Large residues(leucine…) may
occlude the channel by
forming a tight hydrophobic
ring.
- The pore lined by small polar
residues.
Action potentials integrate the activities of several ion
channels working in concert
+ ions move to right side.
There are concentration
gradients between two sides. Channel + ions are accumulated at right side.
open
Make electrostatic force.
Equilibrium Potential: concentration gradient is balanced by the
electrostatic force resisting the motion of a additional charge.
※ Nernst equation
Veq = -(RT/zF)ln([X]in/[X]out)
= -(2.303)(RT/zF)log10([X]in/[X]out)
R=gas constant
F=Faraday constatn
z=charge on the ion X
-Membrane potential at equilibrium is called the equilibrium
potential for a given ion at a given concentration ratio across a
membrane.
-[Na+]in = 14mM [Na+]out = 143mM, equilibrium potential = +62mV
-[K+]in = 157mM [K+]out = 4mM,
equilibrium potential = -98mV
-The resting potential for a typical neuron is -60mV.
Acetylcholine binding
Acetylcholine receptor channel open
(Nonspecific cation channel)
Na+ flow into / K+ flow out = -20mV
Voltage gated Na+ channel open
Na+ flow into rapidly = +30-mV
Na+ channel inactivated
Voltage gated K+ channel open
K+ flow out slowly = -60mV
K+ channel inactivated
13.5 Gap junctions allow ions and small molecules to
flow between communicating cells
※ Gap junction
-Cell-to cell channel,Passageways between the interiors of contiguous
cells.
-Clustered in discrete regions of the plasma membranes.
-20Å central hole.
-The width of the gap between the cytoplasm of the two cells is about
35Å.
-Small hydrophilic molecules( sugars, amino acids, nucleic acids, less
than 1kda) can pass through gap junctions except protein, nucleic acid
and polysaccharides.
-Important for intercellular communication
in some excitable tissues, such as hear muscle.
-A cell-to-cell channel is made of 12
molecules of connexin(30~42kda).
-Connexin : 4 membrane spanning
heices.
-6 connexin are hexagonally arrayed
to form a half channel = connexon
or hemichannle.
- 2 connexons join end to end in the
intercellular space.
The closing of gap junctions by Ca2+ and H+ serves to seal
normal cells from dying neighbors
13.6 Specific channels increase the permeability of
some membranes to water
-Some tissues need to transport water.
-kidney, secretion of saliva and tears.
※ Aquaporin (Peter Agre in red-blood-cell membrane)
-Water channel. 24kda membrane protein
-6 membrane spanning helices.
-Positive residues in the center prevent the transport of protons through
aquaporin; maintain proton gradients