Unit-3

Transport of ions, molecules across

membrane
Relative permeability of a pure phospholipid
bilayer to various molecules
Passive (simple)
diffusion;
Spontaneous,
positive ΔS, negative
ΔG; down or along
concentration
gradient
Hydrophobicity; size;
Partition coefficient K

Membrane potential,
electrochemical gradient
Overview of membrane transport
proteins

Active transport; uphill Facilitated diffusion; water /ions

Non gated and Gated ion
channels
 ATP-powered pumps and Ion channels

1) ATP-powered pumps (or simply pumps) are ATPases ; Pumps utilize

the energy released by ATP hydrolysis to power movement of
specific ions (red circles) or small molecules against their
electrochemical gradient; active transport

2) Channels proteins permit movement of specific ions (or water)

down their electrochemical gradient; Facilitated diffusion; very
rapid rate; Non gated – always open and Gated ion channels- open
upon response to chemical or electrical signal
 Overview of membrane transport
proteins

Carriers
Uniporter- Glucose and Aminoacids
Cotransporters- antiporters and symporters; coupled reactions; Secondary
active transport
• Transporters (carriers), - three groups, facilitate movement of
specific small molecules or ions

3A- Uniporters transport a single type of molecule down its

concentration gradient via facilitated diffusion; Glucose and
amino acids

Cotransport proteins (symporters, 3B, and antiporters, 3C)

– catalyze the movement of one molecule against its concentration gradient
(black circles), driven by movement of one or more ions down an
electrochemical gradient (red circles). Secondary active transport

• Differences in the mechanisms of transport by these three major

classes of proteins account for their varying rates of solute
movement
 Uniport and passive diffusion - Differences

• Uniport- protein mediated; movement of glucose and other small

hydrophilic molecules
– The rate of facilitated diffusion - far higher than passive diffusion

– Partition coefficient (K) is irrelevant

– Via limited number of uniporter ; maximum transport rate Vmax; each uniporter
is working at its maximal rate.

– Transport is specific; Single or group of closely related specific molecules

Model of uniport transport of glucose by GLUT1

Step 1-4
Step 4-1
 GLUT1 Uniporter Transports Glucose

• Catalyzes the net import of glucose from the extracellular medium

into the cell

Outward facing conformation with a bound

glucose

C is the concentration of Sout; Sin= 0

Micromoles per milliliter of cells per hour

KM= 1.5 mM

KM= 20 mM
• Blood glucose - 5 mM, GLUT1 functions 77 percent of the maximal rate

• GLUT1 and GLUT3- erythrocytes and other cells - take up glucose from
the blood continuously at high rates

• GLUT1- transmembrane helices; serine, threonine, asparagine, and

glutamine- side chains H bond with hydroxyl groups of glucose

• GLUT2- liver and islet β cells - insulin secreting

• GLUT4
– fat and muscle cells; respond to insulin;

– absence of insulin, found in intracellular membranes, not on the plasma

membrane

• GLUT5- transports fructose

ATP-Powered Pumps

ABC (ATP-binding cassette) superfamily

• P-class pumps
– Two identical α-catalytic subunits, phosphorylated as part of the
transport cycle.
– Mostly two smaller β- subunits, present in some of these pumps,
may regulate transport
– Altleast one α- subunit is phosphorylated
– Na+/K+ pump- low cytosolic Na+ and high cytosolic K +
– Ca2+ ATPases- pumps outside; from cytosol into Sacroplasmic
reticulum
– acid-secreting cells- transports protons (H + ions) out of and K +
ions into the cell.
• F-class and V-class ion pumps
– Both Similar; Several transmembrane and cytosolic subunits

– Unrelated to P class

– Transport only protons; No phosphorylation

– ATP hydrolysis- V class- low pH of vacuoles. Lysosomes;

against a proton electrochemical gradient

– F class- ATP synthases- Mitochnodria and chloroplast

 ABC (ATP-binding cassette) superfamily

• Ions, sugars, amino acids, phospholipids,peptides, polysaccharides,

or even proteins
• Four “core” domains (2- transmembrane (T) and 2 ATP- binding
domian (A))
 Operational model of the Ca2+ ATPase in the
SR membrane of skeletal muscle cells

high-energy acyl phosphate bond

Why ?
E1-P and
E2- P

Only one of the two α catalytic subunits of this P-class pump is depicted.
• Step 1: E1 conformation, two Ca2+ ions bind to two high-affinity
binding sites accessible from the cytosolic side and an ATP binds to a
site on the cytosolic surface

• Step 2: The bound ATP is hydrolyzed to ADP in a reaction that

requires Mg2+ , and the liberated phosphate is transferred to a specific
aspartate residue in the protein, forming the high-energy acyl
phosphate bond denoted by E1 ~ P

• Step:3 The protein then undergoes a conformational change that

generates E2, which has two low-affinity Ca2+ -binding sites accessible
to the SR lumen
• Step 4: The free energy of hydrolysis of the aspartyl-phosphate
bond in E1 ~ P is greater than that in E2 ~ P, and this reduction in
free energy of the aspartyl-phosphate bond can be said to power the
E1 to E2 conformational change. Spontaneously Ca2+ dissociate
from the low-affinity sites to enter the SR lumen

• Step 5: Hydrolysis of aspartyl-phosphate bond

• Step 6: Dephosphorylation powers the E2 to E1 conformational

change and E1 is ready to transport Ca2+ ions
Structure of the catalytic subunit of the
muscle Ca2+ ATPase
 Plasma membrane Ca2+ ATPase
• Ca2+ ions- inside cell kept below 0.1 – 0.2 M

• Plasma membrane Ca2+ ATPase

• Regulated by calmodulin; cytosolic Ca2+ binding protein

• Increase in cytosolic Ca2+ ; binding with calmodulin ; triggers

allosteric activation of Ca2+ ATPase

• Rapid exit of Ca2+

 Operational model of the Na+/K+
ATPase in the plasma membrane

α 2β2.
 V-Class H+ ATPases Pump Protons Across
Lysosomal and Vacuolar Membranes
• V-class ATPases transport only H ions.
• Membranes of lysosomes, endosomes,
and plant vacuoles- to acidify lumen
• Lysosomal lumen (pH ≈4.5–5.0)
• 2 domains;
– Cytosolic hydrophilic domain (V1)
– transmembrane domain (V0) with
multiple subunits in each domain.
– Binding and hydrolysis of ATP by
the B subunits
Organelle with only
proton pump and no
Cl- channel ; no
significant change in
the intraluminal pH.

Electrogenic
• Organelle with proton pump and Cl-
channel; no electric potential;
accumulation of H+

• (1) movement of an equal number of

anions (e.g., Cl-) in the same
direction; in lysosomes
• or by (2) movement of equal
numbers of a different cations in the
opposite direction ; lining of the
stomach, which contains a P-class
H+/K+ ATPase that is not
electrogenic and pumps one H+
outward and one K+ inward.
 ABC proteins
• Two transmembrane (T) domains (six membrane-spanning α –
helices) and two cytosolic ATP-binding (A) domains
• A domains-30-40 % homologous sequences
• Some ABC proteins- additional exoplasmic regulatory domain
• Permeases- transport specific amino acids, sugars, vitamins, or
even peptides into the cell
• E. coli histidine permease;
– soluble histidine binding protein is located in the periplasmic space
– Directs to T domain of ABC ; Permeation powere by ATP hydrolysis
• E. coli, the outer membrane contains porins
 Eukaryotic ABC protein
• Multidrug-resistance (MDR) transport protein known as MDR1.
• ATP hydrolysis to export a large variety of drugs
• All four domains of MDR1 are fused into a single 170,000-MW protein.
• 50 different mammalian ABC transport proteins
– the liver, intestines, and kidney—sites where natural toxic and waste
products are removed from the body.
– Substrates- sugars,amino acids, cholesterol, peptides, proteins, toxins, and
xenobiotics
– Transport toxins into bile, intestinal lumen etc
– Evolution- transport drugs
 ABC proteins and transport of lipids

Model of E. coli lipid flippase, an ABC protein homologous to mammalian

MDR1
• MDR1 “flips” a charged substrate molecule from the
cytosolic to the exoplasmic leaflet
• Thermodynamics
• MDR2, a homologous protein
– present in the region of the liver cell

– flip phospholipids from the cytosolic-facing leaflet of the plasma

membrane to the exoplasmic leaflet

– Generate an excess of phospholipids in the exoplasmic leaflet

– these phospholipids then peel off into the bile duct and form an
essential part of the bile.
Flippase model of transport by MDR1
and similar ABC proteins
• Step 1 : The hydrophobic portion (black) of a substrate molecule
moves spontaneously from the cytosolinto the cytosolic-facing
leaflet of the lipid bilayer, while thecharged end (red) remains in the
cytosol.
• Step 2 : The substrate diffuses laterally until encountering and
binding to a site on the MDR1 protein within the bilayer.
• Step 3 : The protein then “flips” the charged substrate molecule
into the exoplasmic leaflet, an energetically unfavorable reaction
powered by the coupled hydrolysis of ATP by the cytosolic domain.
• Steps 4 and 5 : Once in the exoplasmic face, the substrate again can
diffuse laterally in the membrane and ultimately moves into the
aqueous phase on the outside of the cell.
 Nongated Ion Channels and
the Resting Membrane Potential

• Plasma membrane contains channel proteins

• Allow the principal cellular ions (Na+, K+, Ca2+, and Cl-)

• Move through them at different rates down their concentration gradients

• Due to pumps and channels- difference in voltage, or electric potential

• 70 millivolts (negative inside cell)

Selective Movement
of Ions

Na+-channel protein
that accommodate
Na+ ions but exclud
K + and Cl- ions
• Nernst equation

If [Nal]/[Nar] 0.1, a tenfold ratio of concentrations as in then ENa -0.059 V (or -59 mV), with
the right side negative with respect to the left

The magnitude of the membrane electric potential is the same (59 mV for a tenfold
difference in ion concentrations), except that the right side is now positive with respect to the
left
• The plasma membranes of animal cells contain many open K+ channels

• Few open Na+, Cl-, or Ca2+ channels.

• Major ionic movement across the plasma membrane is that of K+ from the inside
outward, powered by the K + concentration gradient

• Excess of negative charge on the inside and creating an excess of positive charge on
the outside.

• Thus the outward flow of K + ions through these channels, called resting K
channels, is the major determinant of the inside-negative membrane potential.

• Opening and closing are not affected by the membrane potential or by small
signaling molecules, these channels are called nongated.
 Structure of resting K+ channel

Four identical subunits each containing two conserved membrane-spanning

helices, S5 and S6 (yellow), and a shorter P, or pore segment (pink). P- near
exoplasmic, connect S5 and S6; non helical turret which lines upper part of
pore; a short helix; and an extended loop that protrudes into the narrowest
part of the pore and forms the ion-selectivity filter
Transmembrane forces acting on Na+
ions.
 Cotransporters
• Structural similarities to uniporters
• operate at equivalent rates
• cyclical conformational changes
• transported molecule and cotransported ion- same direction
(symport); opposite directions- antiport
• cation cotransporter
– Na+/H+ antiporter- exports H+ from cells coupled to the energetically
favorable import of Na+
• anion cotransporter- AE1 anion antiporter protein- one-for-one
exchange of Cl- and HCO3-
 Na+-Linked Symporters
• Import aminoacids and glucose into animal cells against high
concentration gradients
• Two-Na+/one-glucose symporter

F is the Faraday constant and E is the membrane electric potential

ΔG of about -3 kcal per mole of Na

At equilibrium ΔG= 0.

Glucose in/Glucose out ≈30,000

Accumulate a very high concentration of glucose relative to the external concentration

Cotransport by Symporters
and Antiporters

Operational model for the two-Na+/one glucose

symporter.
• Step 1: Simultaneous binding of Na+ and glucose to the
conformation with outward-facing binding sites

• Step 2: generates a second conformation with inward-facing sites

• Step 3: Dissociation of the bound Na+ and glucose into the cytosol
allows the protein to revert to its original outward-facing
conformation

• Step 4: ready to transport additional substrate.

 Na+-Linked Antiporters
• Exports Ca2+ from Cardiac Muscle Cells
• Three-Na + /one- Ca2+ antiporter

• Movement of three Na + ions is required to power the export of one

Ca2+ ion from the cytosol
• A rise in the cytosolic Ca2+ concentration in cardiac muscle triggers
contraction
• Lowering – cytosolic Ca2+ by Na + / Ca2+ antiporter reduces the
strength of heart muscle contraction.
 Several Cotransporters Regulate
Cytosolic pH
• CO2 and water H2CO3 (carbonic acid)
• Dissociate to yield H+; dangerous
• remove some of the “excess” protons
– Na+HCO3-/Cl- antiporter
• imports one Na+ ion down its concentration gradient, together with one
HCO3-, in exchange for export of one Cl- ion against its concentration
gradient. carbonic anhydrase

• OH- combine with H+ to form water

– Na+/H+ antiporter
– couples entry of one Na + ion into the cell down its concentration gradient to
the export of one H + ion.
Cytosolic pH of growing cells is maintained very close to pH 7.4.
Concentration of ions and sucrose by the
plant vacuole
• The vacuolar membrane contains two types of proton pumps
(orange): a V-class H+ ATPase (left) and a pyrophosphate-
hydrolyzing proton pump (right) that differs from all other ion-
transport proteins and probably is unique to plants.
• These pumps generate a low luminal pH as well as an inside positive
electric potential across the vacuolar membrane owing to the inward
pumping of H + ions.
• The inside-positive potential powers the movement of Cl- and NO3-
from the cytosol through separate channel proteins (purple).
• Proton antiporters (green), powered by the H + gradient, accumulate
Na + , Ca2+, and sucrose inside the vacuole.
 Movement of Water

• Osmosis, or osmotic flow

• Osmotic pressure is defined as the hydrostatic pressure required to
stop the net flow of water across a membrane separating solutions of
different compositions
• 0.5 M NaCl solution is actually 0.5 M Na+ ions and 0.5 M Cl - ions
and has the same osmotic pressure as a 1 M solution of glucose or
sucrose.
• Need for water-channel proteins
RBC lysis
• When placed in a hypotonic solution (i.e., one in which the concentration of solutes
is lower than in the cytosol), animal cells swell owing to the osmotic flow of water
inward.

• Conversely, when placed in a hypertonic solution (i.e., one in which the

concentration of solutes is higher than in the cytosol), animal cells shrink as
cytosolic water leaves the cell by osmotic flow.

• Consequently, cultured animal cells must be maintained in an isotonic medium,

which has a solute concentration identical with that of the cell cytosol
 Aquaporins Increase the Water Permeability
of Cell Membranes
• Cell-surface protein; water channel
• Tetramer of identical 28-kDa subunits
• Each subunit contains six membrane-spanning helices that form a
central pore through which water moves
• At its center the 2-nm long water-selective gate, or pore, is only 0.28
nm in diameter, which is only slightly larger than the diameter of a
water molecule
• Conserved hydrophilic amino acid residues whose side-chain and
carbonyl groups extend into the middle of the channel.
• Several water molecules move simultaneously through the channel,
each of which sequentially forms specific hydrogen bonds and
displaces another water molecule downstream
Three pairs of homologous transmembrane helices (A and A', B and
B', and C and C') are oriented in the opposite direction with respect
to the membrane and are connected by two hydrophilic loops
containing short nonmembrane- α spanning helices and conserved
asparagine (N)
residues.
Transcellular transport of glucose from
the intestinal lumen into the blood
• The Na + /K + ATPase in the basolateral surface membrane generates
Na and K concentration gradients.
• The outward movement of K + ions through nongated K channels
(not shown) generates an inside negative membrane potential.
• Both the Na + concentration gradient and the membrane potential are
used to drive the uptake of glucose from the intestinal lumen by the
two-Na + /one glucose symporter located in the apical surface
membrane.
• Glucose leaves the cell via facilitated diffusion catalyzed by
GLUT2, a glucose uniporter located in the basolateral membrane.
Acidification of the stomach lumen by
parietal cells in the gastric lining.
• The apical membrane of parietal cells contains an H + /K + ATPase (a
P-class pump) as well as Cl - and K + channel proteins.
• Note the cyclic K + transport across the apical membrane: K ions are
pumped inward by the H + /K + ATPase and exit via a K + channel.
• The basolateral membrane contains an anion antiporter that
exchanges HCO3 - and Cl - ions.
• The combined operation of these four different transport proteins
and carbonic anhydrase acidifies the stomach lumen while
maintaining the neutral pH and electroneutrality of the cytosol
 Voltage-Gated Ion Channels

• Neurons and specialized muscle cells generate action potential

• arrival of an action potential at the axon terminus causes secretion of

chemicals called neurotransmitters

• These chemicals, in turn, bind to receptors on adjacent cells and

cause changes in the membrane potential of these cells.

• Thus electric signals carry information within a nerve cell, while

chemical signals transmit information from one neuron to another or
from a neuron to a muscle or other target cell.
 Morphology
of two types of mammalian neurons

A multipolar interneuron has profusely branched dendrites, which

receive signals at synapses with several hundred other neurons. A single
long axon that branches laterally at its terminus transmits signals to other
neurons
A motor neuron innervating a muscle cell typically has a single long axon
extending from the cell body to the effector cell. In mammalian motor
neurons, an insulating sheath of myelin usually covers all parts of the axon
except at the nodes of Ranvier
and the axon terminals.
• The cell body - nucleus and is the site of synthesis; neuronal
proteins and membrane synthesis.
• Some proteins are synthesized in dendrites, but none are made in
axons or axon terminals.
• Microtubules move proteins and membranes
• Axons, whose diameter varies from a micrometer to millimeter
(squid)
• Specialized for conduction of action potentials.
• An action potential is a series of sudden changes in the voltage, or
equivalently the electric potential, across the plasma membrane.
• Resting (nonstimulated) state, the electric potential across the axonal
membrane is approximately -60 mV ; resting potential.
• At the peak of an action potential, the membrane potential can be as
much as +50 mV (inside positive), a net change of 110 mV
(depolarization)
An action potential originates at the axon hillock, the junction of the axon and cell body, and is
actively conducted down the axon to the axon terminals, small branches of the axon that form
the synapses, or connections, with other cells

One action potential is generated for about every 4 milliseconds.

 Voltage sensitive
Ca2+
channels open
and an influx of
Ca2+, triggers
fusion of vesicles;
Diffusion of NT
(0.5 mS);
Binding to
receptors

Potential becomes
negative inside
postsynaptic cell

Separates Plasma membrane of presynaptic and

postsynaptic cell
• Action potentials move at speeds up to 100 meters per second.

• Arrival of an action potential at an axon terminal leads to opening of

voltage sensitive Ca2+ channels and an influx of Ca2+, causing a
localized rise in the cytosolic Ca2+ concentration in the axon
terminus.

• The rise in Ca2+ in turn triggers fusion of small vesicles containing

neurotransmitters with the plasma membrane, releasing
neurotransmitters from this presynaptic cell into the synaptic cleft,
the narrow space separating it from postsynaptic cells
• Most neurons have multiple dendrites, which extend outward from the cell body
and are specialized to receive chemical signals from the axon termini of other
neurons
• Dendrites convert these signals into small electric impulses and conduct them
toward the cell body.
• Neuronal cell bodies can also form synapses and thus receive signals.
• Membrane depolarizations or hyperpolarizations generated in the dendrites or cell
body spread to the axon hillock.
• If the membrane depolarization at that point is great enough, an action potential will
originate and will be actively conducted down the axon.
• Neurons use changes in the membrane potential, the action potentials, to conduct
signals along their length
• Neurotransmitters, to send signals from cell to cell.
Magnitude of the Action Potential Is Close to ENa

Active potential- sequential opening and closing first of voltage-gated Na+

channels and then of voltage-gated K + channels
Sequential Opening and Closing of Voltage-
Gated Na+ and K+ Channels Generate Action
Potentials

Operational model of the voltage-gated Na+ channel.

• Step 1: Four transmembrane domains in the protein contribute to the central pore
through which ions move. Cutaway views depicting three of the four transmembrane
domains.
• Step 2: In the closed, resting state, the voltage-sensing α helices, which have positively
charged side chains every third residue, are attracted to the negative charges on the
cytosolic side of the resting membrane. This keeps the gate segment in a position that
blocks the channel, preventing entry of Na+ ions.
• Step 3: In response to a small depolarization, the voltage-sensing α helices rotate in a
screwlike manner toward the outer membrane surface, causing an immediate
conformational change in the gate segment that opens the channel.
• Step 4: The voltage-sensing α helices rapidly return to the resting position and the
channel-inactivating segment moves into the open channel, preventing passage of further
ions.
• Once the membrane is repolarized, the channel-inactivating segment is displaced from
the channel opening and the gate closes; the protein reverts to the closed, resting state
and can be opened again by depolarization.
 Voltage-Gated K + Channels
• The repolarization of the membrane that occurs during the refractory period is due
largely to opening of voltage-gated K + channels.
• Increased efflux of K+
• Cytosol is negative
• Briefly membrane is hyperpolarized (more negative than resting potential) which is
approaching to EK
• Opening of the voltage-gated K + channels is induced by the large depolarization of
the action potential.
• Unlike voltage-gated Na + channels, most types of voltage-gated K + channels
remain open as long as the membrane is depolarized, and close only when the
membrane potential has returned to an inside-negative value.
• Because the voltage-gated K + channels open slightly after the initial depolarization,
at the height of the action potential, they sometimes are called delayed K channels.
• Eventually all the voltage-gated K + and Na + channels return to their closed resting
state.
• The only open channels in this baseline condition are the nongated K + channels that
generate the resting membrane potential, which soon returns to its usual value
The upward deviations in the current indicate the opening of K+ channels and
movement of K + ions outward (cytosolic to exoplasmic face) across the membrane.

Increasing the membrane depolarization (i.e., the clamping voltage) from -10 mV
to 50 mV increases the probability a channel will open, the time it stays open, and
the amount of electric current (numbers of ions) that pass through it.
• Unidirectional
conduction of an
action potential
due to transient
inactivation of
voltage-gated Na+
channels
 Schematic depictions of the
secondary structures of voltage-gated K+ and
Na + channels.

4 identical; Each 600–

700 amino acids

Monomer; 4
transmembrane
domains (I-IV);
conserved
hydrophobic motif
(H).
• Voltage-sensing S4 α helices move in response to membrane
depolarization
• Movement of the channel-inactivating segment into the open pore
blocks ion flow

Ball-and-chain model
for inactivation of
voltage-gated K +
channels.
 Myelination Increases the Velocity
of Impulse Conduction

• 10-100 m/s
• Stimulate a muscle to contract within 0.01 seconds; by traveling 1 m
length of axon
• Nonmyelinated neurons, the conduction velocity of an action
potential is roughly proportional to the diameter of the axon
Action potential jumps from node to node along the axon.
• Because voltage-gated Na + channels are localized to the axonal
membrane at the nodes of Ranvier, the influx of Na + ions associated
with an action potential can occur only at nodes. When an action
potential is generated at one node (step 1 )
• the excess positive ions in the cytosol, which cannot move outward
across the sheath, diffuse rapidly down the axon, causing sufficient
depolarization at the next node (step 2 )
• To induce an action potential at that node (step 3 ).
• By this mechanism the action potential jumps from node to node
along the axon.
Neurotransmitters

Nucleotides and corresponding nucleosides-

also function as neurotransmitters.
Neurotransmitters Are Transported into Synaptic
Vesicles by H+ Linked Antiport Proteins
• Critical to synapse function
– secretion is tightly coupled to arrival of an action potential at the
axon terminus
– synaptic vesicles are recycled locally to the axon terminus after
fusion with the plasma membrane.
• Unlike most neurotransmitters, acetylcholine is not recycled.
 Signaling at Synapses Usually Is Terminated
by Degradation or Reuptake of Neurotransmitters (NT)

• After release NT has to be removed or destroyed to prevent

continuous stimulation of post synaptic cell

• Diffusion away from cleft- slow

• Acetylcholine degraded by acetylcholinesterase

• Release choline and acetate

• Choline is transported back into the presynaptic axon terminal by a

Na+/choline symporter
Sequential activation of gated ion channels
at a neuromuscular junction

Release of one acetylcholine- containing synaptic vesicle –opening of

about 3000 ion channels in the postsynaptic membrane
• Arrival of an action potential at the terminus of a presynaptic motor neuron
induces opening of voltage-gated Ca2+ channels (step 1 )

• Subsequent release of acetylcholine, which triggers opening of the ligand-

gated acetylcholine receptors in the muscle plasma membrane (step 2 ).

• The resulting influx of Na+ produces a localized depolarization of the

membrane, leading to opening of voltage-gated Na + channels and
generation of an action potential (step 3 ).

• When the spreading depolarization reaches T tubules, it is sensed by

voltage gated Ca2+ channels in the plasma membrane. This leads to opening
of Ca2+ -release channels in the sarcoplasmic reticulum membrane,
releasing stored Ca2+ into the cytosol (step 4 ).

• The resulting rise in cytosolic Ca2+ causes muscle contraction

 Nicotinic
acetylcholine receptor

α2βγδ
• Pentameric receptor- α2βγδ
• 35–40 percent of the residues in any two subunits are similar
• Five fold symmetry, cation channel is a tapered central pore
• Binding sites: 2 Acetylcholine molecules bind ; interface between αδ
and αγ subunits
• Channel is opened within a few microseconds (0.65–0.80 nm in
diameter)
• Allows passage of hydrated Na+ and K+
• Each subunit has M2 α helix (red)- with Asp, Glu
• The two acetylcholine binding sites are located about 3 nm from the
membrane surface.
• The tunnel-like entrance narrows abruptly after a distance of about 6
nm.
 M2 helix

In the closed state, the kink in the center of each M2 helix points inward,
constricting the passageway, whose perimeter is indicated by the blue spheres. In
the open state, the kinks rotate to one side, so that the helices are farther apart. The
green spheres denote the hydroxyl groups of serine (S) and threonine (T) residues in
the center of the M2 helices; in the open state these are parallel to the channel axis
and allow ions to flow.
 Endocytosis
• Christian deDuve in 1963

• Both the ingestion of large particles (such as bacteria) and the

uptake of fluids or macromolecules in small vesicles
– Phagocytosis

– Pinocytosis

– Receptor mediated endocytosis

 Phagocytosis
Bacteria, cell debris, or even intact cells- “cell
eating”

Binding triggers – extension of pseudopodia (actin

based)

Surround and fuse – form large intracellular vesicle

(>0.25 μm) in diameter) called a phagosome.

Phagosome fuse with lysosome- Phagolysosome

Upon maturation some internalized membrane

proteins- recycled back

Professional phagocytes- Macrophages and

neutrophils
 Pinocytosis

• Cell drinking
• Small droplets of
extracellular fluid
and any material
dissolved in it are
nonspecifically
taken up
• Bulk phase
endocytosis
 Receptor mediated endocytosis

• Receptor ligand interaction

• Selective and efficient uptake of macromolecules

• Clathrin-mediated RME- coated pits in plasma membrane

• Receptors -10-20 times more concentrated at pits

• Network of polygons (hexagons and pentagons)
 Clathrin molecule
• Three heavy chains and three light chains, joined together at the
center to form a three-legged assembly called a triskelion
 Molecular organization of a coated
• Surface of a coated
vesicle
vesicle showing the
arrangement of
triskelions and adaptors
in the outer clathrin coat.
• The sides of the polygons
are formed by parts of the
legs of overlapping
triskelions.
• The N-terminus of each
clathrin heavy chain
forms a “hook” that
projects toward the
surface of the membrane
where it engages an
adaptor
• Cross section through the
surface of a coated vesicle
showing the interactions of the
AP2 adaptor complexes with
both the clathrin coat and
membrane receptors.

• Recruitment of AP2 adaptors to

the plasma membrane is
facilitated by the presence of
PI(4,5)P2 molecules in the inner
(cytosolic) leaflet of the
membrane
Clathrin cage containing 36 triskelions
Phosphoinositides
 The role of dynamin in the formation of clathrin
coated vesicles.
The clathrin lattice of the coated pit (step 1)
undergoes rearrangement to form an
invaginated vesicle connected to the
overlying plasma membrane by a stalk (step
2).
At this point, the dynamin subunits, which
are concentrated in the region, undergo
polymerization to form a ring around the
stalk (step 3).
Changes in the conformation of the ring,
which are thought to be induced by GTP
hydrolysis (step 4), lead to fission of the
coated vesicle from the plasma membrane
and disassembly of the dynamin ring (step
5a).
If vesicle budding occurs in the presence of
GTPγS, a nonhydrolyzable analogue of GTP,
dynamin polymerization continues beyond
formation of a simple collar, producing a
narrow tubule constructed from several turns
of the dynamin helix (step 5b)
The Endocytic Pathway- for internalizing LDL (Low
density Lipoprotein)
• Step 1: Cell-surface LDL receptors bind to an apoB protein embedded in
the phospholipid outer layer of LDL particles. Interaction between the
NPXY sorting signal in the cytosolic tail of the LDL receptor and the AP2
complex incorporates the receptor-ligand complex into forming endocytic
vesicles.

• Step 2: Clathrin-coated pits (or buds) containing receptor-LDL complexes

are pinched off by the same dynamin-mediated mechanism used to form
clathrin/AP1 vesicles on the trans-Golgi network

• Step 3: After the vesicle coat is shed, the uncoated endocytic vesicle (early
endosome) fuses with the late endosome. The acidic pH in this
compartment causes a conformational change in the LDL receptor that
leads to release of the bound LDL particle
• Step 4: The late endosome fuses with the lysosome, and the proteins
and lipids of the free LDL particle are broken down to their
constituent parts by enzymes in the lysosome.

• Step 5 : The LDL receptor recycles to the cell surface where at the
neutral pH of the exterior medium the receptor undergoes a
conformational change so that it can bind another LDL particle
Role of pH
Newly synthesized
lysosomal enzymes (red
spheres)

EGF receptor (shown in

green)

Mannose 6-phosphate
receptors (MPRs)
• The movement of materials from the extracellular space to early endosomes
where sorting occurs.
• Endocytosis of two types of receptor–ligand complexes is shown.
• Housekeeping receptors, such as the LDL receptor (shown in red), are
typically sent back to the plasma membrane, whereas their ligands (purple
spheres) are transferred to late endosomes.
• Signaling receptors, such as the EGF receptor (shown in green), are
typically transported to late endosomes along with their ligands (yellow).
• Late endosomes also receive newly synthesized lysosomal enzymes (red
spheres) from the TGN.
• These enzymes are carried by mannose 6-phosphate receptors (MPRs),
which return to the TGN.
• The contents of late endosomes are transferred to lysosomes by a number
of routes (not shown).
• The inset on the left shows an enlarged view of a portion of a late
endosome with an intraluminal vesicle budding inward from the outer
membrane. The membranes of these vesicles contain receptors to be
degraded.
 Entry of HIV
• Human Immuno deficiency Virus (HIV) – Enveloped retrovirus, Single
stranded RNA
• Attacks and destroys the CD4 cells (CD4 T lymphocyte) of the immune
system
• CD4+- Th (T helper cells)
• HIV uses the machinery of the CD4 cells to multiply and spread throughout
the body.
• Seven steps or stages, is called the HIV life cycle.
– 1) binding, 2) fusion, 3) reverse transcription, 4) integration, 5) replication, 6)
assembly, and 7) budding.
Structure of HIV
https://www.niaid.nih.gov/diseases-conditions/hiv-replication-cycle
Model of the common mechanism for formation of
multivesicular endosomes and budding
of HIV from the plasma membrane

Ubiquitin-tagged
peripheral membrane
protein of the endosome,
known as Hrs

Ubiquitin-binding protein Tsg101

• Bottom: In endosomal budding, ubiquitinated Hrs on the endosomal
membrane directs loading of specific membrane cargo proteins
(blue) into vesicle buds and then recruits cytosolic ESCRT
complexes to the membrane (step 1 ).
• Note that both Hrs and the recruited cargo proteins are tagged with
ubiquitin. After the set of bound ESCRT complexes mediate
membrane fusion and pinching off of the completed vesicle (step 2),
they are disasssembled by the ATPase Vps4 and returned to the
cytosol (step 3).
• Top: Budding of HIV particles from HIV-infected cells occurs by a
similar mechanism using the virally encoded Gag protein and
cellular ESCRT complexes and Vps4 (steps 4 –6 ).
• Ubiquitinated Gag near a budding particle functions like Hrs.

ESCRT- Endosomal sorting complexes required for transport

 Role of Gag proteins

• Budding of virus from infected cell is driven by viral gag protein;

polymerize , spherical shell; vesicle

• Protrude outward from plasma membrane

• N-terminal- Gag protein; associates with plasma membrane

• C-terminal segment - for pinching off of complete HIV particles.

• Gag protein mimics the function of Hrs, redirecting ESCRT (Endosomal

sorting complexes required for transport) complexes to the plasma
membrane where they can function in the budding of virus particles
 Exocytosis

• The fusion of a secretory vesicle or secretory granule with the

plasma membrane and subsequent discharge of its contents is called
exocytosis

• Materials delivered to extracellular space

• Regulated secretion, most notably the release of neurotransmitters

into the synaptic cleft.

• Membrane fusion produces an opening through which the contents

of the vesicle or granule are released into the extracellular space.

• Release of NT?
• Fusion is regulated by a calcium-binding protein (synaptotagmin)
present in the membrane of the synaptic vesicle
• Contact between the vesicle and plasma membranes is thought to
lead to the formation of a small, protein-lined “fusion pore”
• Some fusion pores may simply re-close, but in most cases, the pore
rapidly dilates to form an opening for discharge of the vesicle
contents
• After fusion, luminal surface of vesicle- part of PM and cytosolic
surface of vesicle- part of cytosol
Constitutive- regular secretion, lipids, proteins , waste
Regulated- Secretory cells (Hormone, NT, digestive enzymes) ;
triggered by extracellular ligand
Fusion of vesicles with lysosomes
 Steps
• Four steps in constitutive exocytosis

• Five steps in regulated exocytosis.

• Vesicle trafficking, tethering, docking, priming, and fusing.

• Trafficking: Transport of vesicles (MT and motor proteins).

• Tethering: Vesicle is linked to membrane; pulled

• Docking: Attachment of the vesicle membrane with the cell

membrane. Merge of PM and membrane of vesicle
• Priming: Priming occurs in regulated exocytosis and not in
constitutive exocytosis. Modifications related to signaling

• Fusion:

• Complete fusion- forms fusion pore; contents released; ATP

required to separate vesicle

• Temporary fusion- to create a fusion pore and release its

contents to the exterior of the cell. The vesicle then pulls away
and reforms
 Leading to membrane fusion, a v-SNARE in the vesicle
2 types of tethering proteins membrane interacts with the t-SNAREs in the target
are depicted: Highly membrane to form a four stranded α-helical bundle that
elongated fibrous proteins brings the two membranes into intimate contact (SNAP-25,
(e.g., golgins and EEA1) and one of the t-SNAREs, is a peripheral membrane protein
multiprotein complexes (e.g., that is bound to the lipid bilayer by a lipid anchor rather
the exocyst and TRAPPI). than a transmembrane domain. SNAP-25 contributes two
helices to the four-helix SNARE bundle.
 Four-stranded bundles comprising helices
donated by syntaxin (red), synaptobrevin
(blue), and SNAP-25 (green). SNAP-25
contributes two helices and lacks a
transmembrane domain (yellow)

A speculative transition state in the fusion

of the two membranes. A small water-
filled cavity is shown at the center of the
transmembrane helix bundle.

The transmembrane helices that previously

resided in the two separate membranes are
now present in the same bilayer, and a fusion
pore has opened between the vesicle and
target membrane. The neurotransmitter
contents of the vesicle can now be
discharged by exocytosis.