Biological Membranes

Biological membrane is the barriers that separate the cellular content of the cell or that
of an organelle from its environment. This barrier is essential for the cell/organelle to
sustain life and maintain its identity. Biological membranes are not just inert barriers
but are dynamic, semi permeable and have a number of biochemical and physiological
functions.
There are two types of biological membranes depending on their location in the cell.
I. Plasma membranes or cell membranes
This is present in all cells of the prokaryotes and eukaryotes and encloses the cellular
contents and defines the boundary of the cell.
II. Intracellular membranes or internal membranous structure
Eukaryotes contain extensive intracellular membranes that segregate specific regions
from the cytoplasm- the sub cellular organelles. These intracellular membranes separate
the cells into compartments with an identity of their own to carry out specific functions.
However, they remain in contact with the cytoplasm of the cell through specific
proteins in these membranes. The plasma membrane is ~10% of the total membranes in
the eukaryotic cells.
These inter cellular membranes in animal cells include those of nucleus, endoplasmic
reticulum, golgi apparatus, mitochondria, lysosomes, peroxisomes and vacuoles. Plant
cells, in addition to the above, have those of choloroplasts.
Although prokaryotes lack these intracellular membrane systems, their plasma
membrane may be in folded to form structures known as mesosomes. The
physiological/biochemical functions associated with the intracellular membranes of
eukaryotic organelles are performed by plasma membranes in prokaryotes. However,
the photosynthetic bacteria contain internal membrane around the vesicles that contain
proteins and photosynthetic assemblies involved in light reactions and initial steps of
photosynthesis.
Besides these membranes, most cells synthesize and secrete coats of one kind or
another that are external to the cell membrane - the extra cellular or external
membranous structures/surfaces. These include: cell walls, calyx, fuzzy coat and
desmosomes, tight junctions, etc. present in the prokaryotic, plant and some animal
cells. These have a specialized structure and a supportive role.
Important Functions carried out by Plasma and Intracellular Membranes
Different biological membranes are associated with various functions. To innumerate:
• Permit and in some cases enhance the absorption of essential nutrients into the cell while
preventing the diffusion of needed metabolites e.g. Biological Transport, absorption
through intestinal epithelial cells and endocytosis.
Secrete cellular products eg. excretion through kidney epithelial cells.
• Keep out toxic material.
• Carry out exocytosis and endocytosis.
• Maintain intracellular pH, ionic concentration
• Carry out energy transduction processes such as oxidative phosphorylation by mitochondial
inner membrane and photophosphorylation by chloroplast thylakoid membranes.
• Control the flow of information between the cells - Signal Transduction - via specific receptors
for hormones, neurotransmitters and other cell signals. eg.. Role in nerve impulse
transmission, muscle contraction, hormone action etc.
• Hold the cells together by cell: cell interactions.
• Role in cell recognition and adhesion.
• Bind certain cellular constituents particularly enzymes/ proteins in an advantageous location
for their specific biochemical functions e.g. Electron transport carriers in mitochondrial and
thylakord membranes, transport proteins in plasma membrane and specific orientation of
membranes enzymes.
• Compartmentation enables diverse metabolic processes, many of which are incompatible
with one another to occur in the cell and thereby prevent futile metabolic cycle e.g.
glycolysis (break down of glucose to pyruvate) occurs in cytosol whereas specific
gluconeogenic enzymes are located in mitochondria and lysosomes. Thus, Hexokinase, a
soluble enzyme, is involved in phosporylating glucose to glucose-6-phosphate and glucose
phosphatase hydrolyzing glucose-6-phosphate to glucose is present in lysosomes.
The supportive, semi permeability, transport (by transport proteins, channels and
pumps), compartmentation roles, signal transduction (through receptor proteins), cell:
cell recognition and adhesion, exocytosis and endocytosis, essential to the physiological
functions of the cell, indicate a complex and a dynamic nature of the biological
membranes.

Common Features of Biological Membranes

In spite of such diversity in function, the biological membranes have a number of
common characteristic features. They are:
– Few molecules thick (6-10nm),

– Bimolecular sheets with lipids forming a bilayer,

– Closed boundary,

– Dynamic,

– Cooperative,
Lipid -protein assemblies held together by noncovalent interactions.

– Self annealing,

– Asymmetric,

Each of the lipid bilayer is referred to as a monolayer or leaflet; the exterior is known as
the exoplasmic face and the interior as the cytoplasmic face of the membrane.
Membranes, also, contain varying amounts of carbohydrates (0 to 10%) linked
covalently to lipids as glycolipids or to proteins as glycoproteins.
Membranes differ in composition and presence of specific proteins which are
responsible for their specific functions.
Models for the Structure of Biological Membranes
Early Models In 1917, Langmuir first made artificial membranes using phospholipids
dissolved in benzene. In 1925, E.Gorter and F. Grendal (on the basis of studies on
erythrocyte membranes) first said that in membranes phospholipds are arranged in a
bilayer which is stable).

In a subsequent model of membrane structure, J. Danielli and H .Davson in 1935,

proposed a Lipid bilayer separated by “Lipoid” material and globular proteins present
at the two surfaces of the lipids.(Fig. a) This was further adapted by J.D.Robertson in
1959, who gave the “Unit membrane” model (Fig. b) which showed the lipids arranged
as a single bilayer with protein in an extended conformation on the outside at the
hydrophilic heads on each side of the bilayer. In this model, the integrity of the
membrane was ‘lipid derived’. Another model proposed by Benson’ postulated the
membrane integrity to be ‘protein derived’ with lipid bilayers interspersed in the
proteins.

Limitations

However, these models could not explain all the characteristics and properties of
membranes
Fluid-Mosaic Model
In 1972, J.S .Singer and G.I Nicolson gave the Fluid-Mosaic model for the membrane
structure.
The fluid mosaic model proposed in 1972 by S. Jonathan Singer and Garth L. Nicolson
for describing the arrangement of lipid and protein within a membrane.
According to the fluid mosaic model, the membrane is a dynamic structure in which
both proteins and lipids can rapidly and randomly diffuse laterally or rotate within the
bilayer. Membrane proteins are visualized as icebergs floating in a highly fluid lipid-
bilayer sea (Actually, some proteins are immobile, and some lipids have restricted
movement.)
Essentially, it suggests that the phospholipid molecule is the repeating unit in a bilayer
arrangement with a thickness of 5-10 nm
Also, the model incorporated the idea that the membrane is a dynamic and not a static
structure with lipids and proteins capable of lateral diffusion in the plane of the bilayer
unless restricted by specific interactions.
The model successfully explained the permeability barrier properties.

Membrane Lipid Rafts and Caveolae

Membrane rafts are regions of the membrane bilayer enriched in sphingolipids, sterols,
and certain proteins. Some integral membrane proteins can be tightly bound to a layer
of phospholipids to form boundary lipids or microdomains. In some plasma
membranes, sphingomyelin and cholesterol interact with and bind membrane proteins
to form lipid rafts. They are cholesterol-sphingolipid microdomains in the outer
monolayer of the plasma membrane

These lipid rafts are remarkably enriched in two classes of integral membrane proteins:
those anchored to the membrane by two covalently attached long-chain saturated fatty
acids (two palmitoyl groups or a palmitoyl and a myristoyl group) and GPl-anchored
proteins

The lipid rafts help to bind proteins in a specific orientation and coordinate and
regulate a variety of signaling processes. Certain proteins such as GPI anchored
proteins, some of Receptor Tyrosine kinases belonging to src protein family or some
proteins after activation such as β-cell receptors and T-cell receptors are associated with
lipid rafts.

Lipid rafts are implicated in cell signaling, molecular trafficking and in certain diseases
such as HIV and malaria.
Caveolae are small (50-100 nm) flask shaped structures which are rich in proteins i.e
invaginations of the plasma membrane in many cell types such as endothelial cells and
adipocytes.

Caveolin is an integral membrane protein with two globular domains connected by a

hairpin-shaped hydrophobic domain, which binds the protein to the cytoplasmic leaflet
of the plasma membrane. Three palmitoyl groups attached to the carboxyl-terminal
globular domain further anchor it to the membrane. Caveolin binds cholesterol in the
membrane, and the presence of caveolin forces the associated lipid bilayer to curve
inward, forming caveolae ("little caves") in the surface of the cell. The protein, caveolin,
has a cytoplasmic C-terminus and a cytoplasmic N-terminus, linked together with
hydrophobic hairpin that is inserted into the cytoplasmic leaflet of the membrane which
results in a change in the morphology of the membrane.

Caveolae are unusual rafts: they involve both leaflets of the bilayer the cytoplasmic
leaflet, from which the caveolin globular domains project, and the extracellular leaflet, a
typical sphingolipid/cholesterol raft with associated GPl-anchored proteins. They are
actually a type of lipid rafts containing the protein caveolin along with other proteins.
They have several functions membrane trafficking within cells and in signal
transduction of external signals into cellular responses. The receptors for insulin and
other growth factors, as well as certain GTP-binding proteins and protein kinases
associated with transmembrane signaling. They also play a role in endocytosis and
oncogenesis.
Plasma Membranes at Intercellular Junctions
In animals and plants, cells within a tissue or an organ are tightly packed. However
adjacent cells often allow direct contacts of various types i.e. they adhere, interact and
communicate through contacts between their plasma membranes. Thus,
plasmodesmata in plant cell walls and tight junctions, desmosomes and gap junctions in
animals are the main types of intercellular junction. Tight junctions and desmosomes
are absent in plants.
Plasmodesmata
Plasmodesmata (singular, plasmodesma) are channels between the plant cells
connecting cytoplasm of adjacent cells (Fig. d). Their diameter is 30 to 60 nm and appear
to be lined by the cell membrane i.e. the plasma membranes of adjacent cells are
continuous through plasmodesmata in the channel. Water and small solutes can pass
freely from cell to cell and probably RNA molecules can also pass by moving along
fibers of the cytoskeleton.
Gap Junctions
Adjacent cells have channels which are cytoplasmic connections. These channels are
2nm in diameters. Gap junction is a hexamer and the subunits are arranged in a rosette
structure. The two hexamers of adjacent cells juxtaposed to from a channel (Fig. 30 a).
This permits the passage of ions or small molecules such as sugars, amino acids etc
between the cells .The calcium ion concentration in the cells regulate the
opening/closing of the gap junctions by tilting of the hexameric subunits (Fig. 30b).
Thus, the metabolic activities of the cells in the tissue can be regulated by passage of
signaling molecules such as cyclic AMP, Ca++ etc.
Tight Junctions
The bands of plasma membranes proteins of adjacent cells are fused in a tight junction
(Fig. 30a) to form a seal around each cell in a layer of tissue and prevent leakage of
nutrients between cells and across the cells e.g. the tight junctions around intestinal
epithelial cells and those in pancreatic acini prevent leakage of pancreatic proteins and
digestive enzymes from central cavities into the blood. Tight junctions occur only in
vertebrates while septate junctions perform the same function in invertebrates and are
slightly different structures
Desmosomes
Desmosomes consist of plaques of dense fibrous material between cells. They fasten
cells into strong epithelial sheets. Clusters of filaments from the cytoplasm of adjacent
cells loop in and out of them. They give tissues mechanical strength and rigidity to the
tissue (Fig. 30a).

Extra Cellular Membranous Structures

As mentioned earlier, the membranous structures, external to the plasma membrane of
the cell include cell walls, extra cellular matrix, calyx or fuzzy coal, tight junctions and
desmosomes etc.
Plant Cell Walls
The distinguishing feature between a plant and an animal cell is the presence of cell
walls around the plant cells. The cell walls protect the cell, maintain its shape, provide
rigidity and prevents excessive uptake of water. These strong cell walls enable the plant
to stand upright. These cell walls are much thicker than the plasma membrane and the
thickness ranges from 0.1 um to several micrometers.
The exact chemical composition of the wall varies from species to species and from one
cell type to another in the same plant. However, the basic design of the cell wall is
consistent. The basic architecture involves ground substance or matrix consisting of
polysaccharides and proteins in which the microfibrils made of polysaccharide cellulose
are embedded. Hemi cellulose, a highly branched polysaccharide made up of 50ß (1→4)
linked sugars of a single type are hydrogen bonded to n cellulose microfibrils and helps
to bind microfibrils to each other and to other components of the matrix . One of these is
pectin which is crosslinked to hemicellulose to form a complex network present in the
principal cell wall components and binds adjacent cells together. Major protein,
extensin, is a glycoprotein rich in serine–hydroxy proline -hydroxy proline–
hydroxyproline sequences which are glycosylated .
Another component is lignin complex, an insoluble polymer of phenolic residues. This
is present in all cell walls and is the strengthening material. The cell walls are highly
impermeable to diffusion of particles with diameter greater than ~4 nm but water and
ions can diffuse freely in cell walls.
A young plant cell first secretes a thin and flexible wall - the primary cell wall (Fig. 29).
Between primary walls of adjacent cells is the middle lamella, a thin layer rich in sticky
polysaccharides called pectin. When the cell mature and stops growing, it strengthens
its wall by secreting hardening substances into the primary wall of some plant cell ,
while others add a secondary cell wall between the plasma membrane and the primary
wall which makes the cell strong and protects it. Wood consists mainly of secondary
walls.
Bacterial Cell Walls
All prokaryotes contain a cell wall which is external to the plasma membrane or cell
membrane. The cell wall maintains the shape of the cell protects the animal and
prevents lysis in hypotonic environment. However they can plasmolyze and die in
hypertonic environment. This is the basis of preservation of meat and pickles where
high salt concentrating protects from bacterial infections.
These cell walls differ in molecular composition and construction from those of plants
and fungi. The classification of bacteria is on the basis of their cell walls taking up the
gram stain which is a dye-iodine complex. Thus, the gram positive bacteria retain the
stain while the gram negative does not. The cell walls of the gram positive bacteria are
simple and contain large amount of peptidoglycan while those of gram negative have a
more complex structure, less peptidoglycan and an outer lipid bilayer membrane and
also contain lipoproteins and lipopolysaccharides) which are more toxic, protect the
bacteria from the defenses of the host and render them more resistant to antibiotics than
the gram positive bacteria.
Gram-positive Bacteria
Their cell wall consists of a thick peptidoglycan layer made up of alternating N-acetyl
glucosamine and N-acetyl muramic acid polysaccharide chains and short peptides
linked by glycine pentapeptides. The short peptides (tetra peptides) have unusual
structure with the sequence L-ala-D-glu-L-lys-D-ala and having some D–amino acids
and the linkage of glutamic acid in the chain is via its γ-carboxyl group. The ε-NH2
group of each lysine is linked to a glycine pentapeptide which is bonded at the other
end to the terminal D-Ala residue of an adjacent chain.
Biological Transport
The lipid bilayer of the membranes is permeable to only hydrophobic molecules and
small uncharged moelcules such as CO2, N2, O2, NO, Urea, ethanol, anaesthetics and to

some extent water by simple diffusion. However, the lipid bilayer is impermeable to
+, K+, Ca2+, Cl-1, HPO 2–, HCO –, Glucose, and
most ions, polar molecules such as Na 4 3

other sugars, amino acids, ADP3–, ATP4– and other organic molecules of biological
importance. These ions and hydrophilic organic molecules are required by all living cells to carry
out cellular activities and meet their metabolic requirements.

Living cells have, thus, evolved systems which enable them to transport ions and
molecules of interest into and out of the cell and intracellular organelles. The biological
transport is mediated by transmembrane integral proteins which are of three types (Fig.
below):
(I) Passive Transporters, which carries out facilitated or passive biological transport.
(II) Active transporters or pumps, which carry out active transport.
(III) Channels.

Passive and Active transporters are also termed as mediators or Facilitators. They have
specific binding sites for the molecules or the solute to be transported and are saturable
and show other characteristics discussed below.
Channels are usually less specific or non specific and do not have a specific binding site
for the solute / molecule which flows through the channel passively i.e. down the
concentration gradient.
On the other hand, ionophores (ion bearing) are antibiotics, natural or synthetic, low
molecular weight compounds that mediate ion transport down an electrochemical
gradient. They are either mobile carriers or form channels. They have been widely used
as models systems to study membrane functions.
There are three general types of Transport.
1. A Uniport is the transport of a single molecule at a time. The transport by glucose
carrier of erythrocyte is a uniport.
Co–transport is the transport of two different types of molecules at the same time.
2. When two different molecules are transported in the same direction, the transport is
symport e.g. Na+ glucose transporter of intestinal epithelial cells.
3. When the two different molecules are transported in opposite directions, the

transport is antiport e.g. Na+/K+ ATPase and ADP /ATP transporter.

3- 4-

The transport can be:

1. Electroneutral if there is a charge neutralization by symport of oppositely charged

ions or antiport of similarly charged ions. e.g. (H – K ) – ATPase of gastric mucosal

+ +

parietal cells.

2. Electrogenie if a charge separation results during transport. Thus, ADP /ATP

3- 4-

Transporter is an electrogenic antiporter.

1. Passive transport
• Simple Passive Diffusion or Transport
• Facilitated Passive Transport
2. Active transport
• Primary active transport
• Secondary active transport
3. Group Translocase
Model for the Mechanism of Biological Transport
The exact mechanism for biological transport is not yet elucidated. Since the integral
membrane transport or facilitator proteins can neither rotate, diffuse nor flip-flop
within the lipid bilayer of the membrane. It is postulated that these proteins undergo
conformational changes during transport, where the solute to be transported binds to
the transporter specifically on one side of the membrane and is released on the other
side.
Thus,
1. The protein must at least exist in 2 states A and B.
2. In state A, it has a high affinity binding site for the specific solute molecule facing one
side of the membrane.
3. Binding of the solute molecule to the protein in state A changes it to the
conformational state B.
4. In state B, the binding site faces the other side of the membrane and has little or no
affinity for the solute, with the result that the solute is released to the other side.
5. on dissociation of the solute, the transporter switches back to the original
conformation State A.
This alternate access and release mechanism has been postulated for major facilitator
transport proteins where solute acquisition or release provides the requisite energy and
controls the conversion of one conformational state to another.

In case of Active Transport or pumps, the solute binding in conformational state A

leads to the phosphorylation of a specific residue on the protein by ATP which leads to
a change in conformation to State B when the solute is released to the other side.
Dephosphorylation reverts the conformation to State A; Such a model has been
postulated for Na+ / K+ ATPase an antiport where Na+ is pumped out of and K+ is
pumped into the cell.
Facilitated Passive transport
When transport of biological membranes is studied using purified membranes protein,
certain common features are observed.
 • The transport of the solute / molecule can be in either direction depending on
the concentration gradient of the solute, from a higher to a lower concentration,
i.e. there is a down hill movement.
• The transport is highly specific. The specificity for a particular transport system
is akin to that of enzyme for its substrate. Thus, the transport system for D–
glucose will not transport any other sugar or at a very low rate.
• The transport is not only specific for a particular molecule, it also shows
stereospecificity i.e. can differentiate between D– and L– Sugars or between L– and
D– aminoacids etc.
• The transport shows saturation kinetics i.e. the rate of transport reaches a
maximum as the concentration of the solute is increased till a maximum
transport rate is reached. This distinguishes biological transport from simple
diffusion or unfacilitated transport where the rate is directly proportional to
concentration gradient .
• The transport is inhibited by known protein reagents that react with specific
groups of proteins as in the case of enzymes e.g. p–chloromericuribenzoate or other
mercurials that react with –SH groups (cysteine residue); fluorodi nitrobenzene, FDNB,
which reacts with –NH2 group etc.

Kinetics of (a) Passive Transport and (b) Facilitated Transport (Source: Zubay, G.
Biochemistry, 1984)
The kinetics of facilitated passive transport are similar to that of enzyme kinetics
exhibiting maximum transport rate viz. Vmax for enzymes and a high affinity binding
site for the solute transported across biological membranes.
These characteristics showed the involvement of proteins with specific binding sites
and that the transport is not by simple passive diffusion but is facilitated by proteins.
These proteins have been called as mediators, carriers, porters, transporters by different
workers as they were studied and the transport system as Facilitated Passive Transport.
However, presently it is referred to as Passive transport or Facilitated transport carried
out by transport proteins or transporters. In some cases the original names have been
retained such as Anion channel or Cl–/HCO exchanger, glucose carriers etc.
Examples
Anion Channel or Exchanger or Cl–HCO–3 Exchange Protein
-
This is a 89–KD dimeric protein which traverses the membrane 12 times. It is an Cl
/HCO - electro neutral antiport). Its N–terminal domain extends towards the cytosol
3

and is associated with the peripheral protein, ankyrin which binds it to the

cytoskeleton.

There is a one to one exchange of HCO3 for Cl .Transport of HCO3 out of the cell is
- - -

coupled to inward transport of Cl-. Carbon dioxide accumulated in the erythrocytes

from respiring tissues is rapidly diffused out of the red blood cells in the lung

capillaries (Fig. 42). Its role is to increase the CO

2 carrying capacity of the blood.
2.
Glucose Transporter (GLUT–1)
Glucose is a universal energy source and is taken up by a variety of transport proteins
by different types of cells.
Glucose Transporter (GLUT–1) of erythrocyte is a 55–KD glycoprotein.

Glucose transporter is a uniport, transporting glucose in either direction down the

concentration gradient. It thus helps to maintain glucose concentration (levels) in the
blood.
Various glucose transporters, depending on their locations in tissues, are known in
humans. Thus,
GLUT–1 is present in most tissues
GLUT–2 mostly in pancreatic B cells while
GLUT–4 in muscle and fat cells which are responsive to insulin.
Active Transport
This type of transport is also carried out by transporters and is referred to as PUMPS.
Active transport shows similarity to Passive Transport in
1. Saturation kinetics.
2. Specificity
 3. Stereospecificity
4. Inhibition by specific protein reagents.
However, they differ
1. in the movement of the solute across the biological membranes which is
(a) unidirectional and in a specific direction and
(b) against the concentration gradient or uphill.
2. and require an energy input i.e. are accompanied by hydrolysis of ATP or any other
source of energy for the uphill movement of the solute and hence named as Pumps.
3. are inhibited by cyanide and such reagents that inhibit energy production.

Primary Active Transport The type of active transport where ATP is the source of energy
is known as Primary Active Transport. Examples of active transport are Na+/K+
ATPase or calcium pumps.
(i) ATP as energy source
(a) P–type ATPases

e.g. Na – K ATPase, Ca – ATPase, H – ATPase

+ + 2+ +

(b) F–type ATPases

eg. F1–F0 ATPase of mitochondrial inner membrane.

F1– F0 ATPase of chloroplast thylakoid membrane.

(c) V–type ATPase in plant vacoules.

(d) A–ATPase in bacteria
(ii) Respiratory electron transport chains which pump H+ out of mitochondrial matrix.
(iii) Light driven H+ pumps of Halobacterium, bacteriorhodopsin and in
photosynthesis.
Secondary Active Transport
2-
(a) H+ – PO4 – antiport in mitochendria

(b) H – K antiport in mitochendria

+ +
(c) H – lactose symport in bacteria
+

(d) Na – glucose symport in intestinal and kidney epithelial cells

+

(e) Na – aminoacid symport in intestinal and kidney epithelial cells

+

(f) Na – Ca Antiport in heart muscle

+ 2+

In this case, the solute is transported uphill coupled to the downhill transport of another
different solute which has been originally pumped uphill by the Primary Active
Transport. Thus, a concentration gradient of Na+ maintained by Na+/K+ ATPase or a
proton gradient is used to transport a second molecule. Examples are Na+–glucose
transport and H+ Lactose transport. Some cells may utilize 30% to 50% of ATP on active
transport.

Two types of Active transport (a) Primary Active Transport, (b) Secondary Active
Transport (Source: Nelson, D.L and Cox, M. Lehninger Principles of Biochemistry, 2005)
Examples of active transport:
Na+ – K+ ATPase or Sodium Pump of Plasma Membranes

The classical example of Primary Active Transport is Na –K ATPase, also, known as

+ +

sodium pump and is the most thoroughly studied active transport system.
It is (αβ) dimer, each consisting of α and β subunits. The 110 KD α–submit contains the
binding sites for the ions and the site phosphorylated by ATP. The 55 KD β–subunit is
glycoprotein whose function is not known.
The α–subunit has around 8 α-helices forming the transmembrane domain and two large
cytoplasmic domains. An aspartyl group in α-subunit is phosphorylated by ATP to
form a reactive aspartyl phosphate intermediate which has been isolated by reduction
with borohydride to homoserine.
The β-subunit has a large extra cellular domain with carbohydrate moieties on the
exterior side and a single Transmembrane helix.

Its known inhibitors cardiotonic steroids and ouabain (wah bane) binds on the external
side, so also the K+, while the Na+ and ATP binding sits are on the cytoplasmic side

The putative dimeric structure of Na+–K+ ATPase (Source: Voet, D and Voet J.
Biochemistry, 1995)

It is called sodium pump as it pumps Na+ out of and K+ into the cell against

It is, thus, an electrogenic antiport as 3Na are pumped out and 2K pumped in. The
+ +

molecular model given for this pump involves two conformational states E
1 and E2
where
1. E1 faces cytoplasmic side and has a high affinity Na+ binding site.
2. Na+ binding to E – [E 3 Na+] promotes phosphorylation of aspartyl residue by ATP
1 1

+]
[E1 – P . 3Na

3. Phosphorphorylation of E1 results in a change of conformation to [E2 – P . 3Na ].

+

4. E2 site faces outside and has a low afinity for Na+ which is released to form [E 2–P]

++.
where the site has a high affinity for K

5. Binding of K forms [E2 – P. 2K ] which results in dephosphorylation of and release of

+ +

Pi into the cytoplasm and reversal of the conformation [E +].

1– 2K

6. E1 having a low affinity for K , releases K in the cytoplasm.

+ +

Significance of Na+ – K+ ATPase

It is responsible for maintaining the intracellular concentrations of Na and K and

+ +

generally transmembrane electrical potential which is essential for electrical excitability

of nerve cells. The sodium gradient is also used to drive the cotransport of several

solutes in many cell types e.g. Na+–glucose and Na+–amino acids symports in intestinal
and kidney epithelial cells, Na+– Ca2+ antiport in muscle cells etc. All cells utilize a large
fraction of the produced ATP (70% in nerve cells) to maintain the required Na + and K+

concentration.

Others include:
• Ca2+ – ATPase or Calcium Pumps
• ATP–Binding Cassette (ABC) Transporters or Multidrug ABC Transproter
Secondary Active Transport
The ion gradients established by primary transport of Na+ or protons can provide
energy for cotransport of other solutes such as another ion, sugar, aminoacids or
dicarboxylic acids.
Na+–Glucose Symport

Na glucose and Na aminoacids cotransport system are present in the specialized

+ +

plasma membranes of intestinal and kidney epithelial cells. The extracellular high

concentration of Na+ drives the symport of glucose and aminoacids by specific

symporters in the apical region of the cells. The Na+ concentration is maintained by the

pumping of Na+ into blood by Na+–K+ ATPase present in the basal region of these cells

The Na –glucose symporter in the epithelial cells of the intestine is a two Na+ - one
+

glucose symporter.

2, Lactose – H+ Symporter
Lactose–H+ symporter is driven by Proton gradient established by proton pump drives the
uphill movement of Lactose
The E.coli lactose transporter has 12 membrane spanning α-helices, the cytoplasmic
domains and the extraperiplasmic domains exhibit two fold symmetry.
The transport of lactose is proposed to be through formation of the two halves of the
transporter which undergo conformational change during one cycle of transport. In one
state the lactose binding site is exposed to the periplasmic. Space where lactose binds. In
other state lactose is released. The interconversion between the two states is due to
protonation in the pairing of charged side chain residues such as Glu 323 and Arg 302
occurring by transmembrane proton gradient.

• Group Translocase
This is a type of Active Transport where phosphoenol pyruvates, PEP, is used by many
bacteria to transport sugars into the cell and also to modify the sugars chemically to
sugar-phosphates. The transport most studied is the phosphoenol pyruvate–glycose
phosphotransferase system, (PEP – PTS) of E. coli. The high energy compound, Phosphoenol
pyruvate (PEP) is the energy source and the sugar is released as sugar-phosphate inside the cell.
The sugar phosphate is retained since the cell membrane is impermeable to them. There are no
transporters for sugar phosphates in the membranes. Some of the PTS–transported sugars are
glucose, mannose, mannitol etc.
This transport involves several soluble and membrane bound proteins catalyzing the
reactions
• Enzyme I - a dimer of 70 KD subunits.
• HPrr (Histidine containing Phosphocarrier protein) - a monomer of 9.5 KD.
• Enzyme II, or mannitol permease, is a monomer of 68 KD
• Enzyme III
Enzyme I and HPrr (Histidine containing Phosphocarrier protein) are soluble proteins
required for all sugars transported. Enzyme II is an integral membrane protein specific
for type of sugar to be transported and requires diacyl glycerol as a cofactor. In E.coli,
there are seven different proteins of Enzyme II, each specific for a sugar molecule. In
some cases, a sugar specific Enzyme III protein is attached loosely to enzyme II – on the
cytoplasmic side of the membrane for some sugars and cytoplasmic for others, is
involved in phosphorylation of sugar as it is transported
It involves four phosphorylated intermediates

PEP → EI → HPr → EIII

glu → EIIglu → glucose

A specific Histidine residue in Enzyme I, HPr and Enzyme III is phosphorylated by PEP
during the transport reaction.
glu is phosphorylated.
A cysteine residue of EII
Channels
These are transmembrane proteins which form channels or pores through which ions
and molecules can flow down their concentration gradient passively depending upon
the pore size and amino acid residues lining the channel. Here, the movement of ions or

water is very fast, 10 to10 s . The channels do not exhibit the characteristics of
7 9 -1

biological transporters i.e. specific binding and saturation kinetics.

Examples of channels include;

• Ion Channels
• Gap junctions
• Aquaporins or water channels
• Bacterial porins
• Ion–Channels
Gated ion-channels are multimeric proteins which open in response to a specific signal
such as
 • voltage or membrane potential changes and
• ligands such as neurotransmitters and
• Signal gated c–AMP and c–GMP

Voltage gated, Na –Channels and K Channels are present in brain and nervous tissues.
+ +

The voltage gated Ca2+ channels are present in muscle, heart and neuromuscular

junction where they have a role in muscle contraction and release of acetylcholine from

vesicles.

Ligand gated channels are the neutransmitter receptors which open on binding of a
specific ligand such as Acetylcholine, Gama-amino butyric acid, GABA. They are
present in brain and nerve tissues. They have an important role in conduction of nerve
impulse and maintaining ion gradient in the nervous tissue; acetylcholine receptor is a
cation channel whereas GABA receptor is an anion channel.
The c–GMP and c–AMP gated ion channels are abundant in the sensing cells and have a role in
visual and olfactory systems.
• Gap Junction
Gap junctions are present in the plasma membrane connecting the adjacent cells in
several tissues. These are hexameric protein channels which appear as ‘Rosettes’.
Molecules upto 600D such as glucose, ATP, ions and other small molecules can diffuse
through these channels to regulate metabolic activities between the cells. The opening
and closing of these gap junctions is regulated by Ca concentration which induces a
2+

conformational change by tilling of the hexameric units

• Aquaporins or Water Channels

Aquaporins are a family of integral membrane proteins which act as specific water
channels. They are involved in rapid movement of water molecules across all plasma
membrane in a number of animals and plant tissues and in bacteria and were
discovered by Peter Agre. Ten Aquaporins are known in human such as in
erythrocytes, renal medulla, nephrons etc. Aquaporin I (API) plays a central role in
water reabsorption of kidney.
Human Aquaporin exists as a homotetramer homologous protein in the
plasmamebrane. It has a unique fold referred to as “hour glass” or “dumb bell”. The
protein contains four pores, one per subunit. Each 28 KD monomer contains four
transmembrane α-helices.
• Bacterial Porins
Porins are present in outer membranes of bacteria. Also, mitochondra and chloroplast
outer membrane contains pores which are nonspecific and allow movement of certain
small molecules to pass through.
Exocytosis and Endocytosis
Cells release or take up large molecules or material in a controlled mechanism by
forming membrane enclosed vacoules or vesicles around them in a process known as
Exocytosis or Endocytosis respectively. The flexibility and self-sealing properties of
biological membranes as also the fusion of membranes mediated by specific proteins
play an important role in exocytosis and endocytosis and viral invasion. Farmation of
vesicles and their transport within the cell plays on important role in these processes.
These vesicles are targeted to different destinations where active guided transport of
vesicles may be involved Microtubules and associated ATP driven motors appear to
have a role in the intracellular transport of vesicles. Molecular motor, kinesin and
dynein, transport vesicles along microtubules in the axon of a nerve cell.
Exocytosis
This is the process where the cells releases large molecules such as secretory proteins
and waste products contained in a membrane vesicle or vacoules to the outside of the
cells or to the plasma membrane. The latter provides a mechanism to form and remodel
plasma membrane i.e. deliver membrane proteins (receptors, enzymes etc.) and lipids to
the plasma membrane. Exocytosis is a common secretory mechanism in eukaryotes.
Exocytosis is of two types:
I. Constitutive Exocytosis II. Regulated Exocytosis
Constitutive Exocytosis

This type is independent of Ca and other signals and is performed by all cells. It serves
2+

to release:

(i) Components to the extracelluar matrix such as carbohydrate chains and proteins
from golgi vesicles to the outside of plant cells during cell wall synthesis.
(ii) Serum proteins from liver.
(iii) Waste products from the cell by protozoa.
(iv) And to delivers newly synthesized membrane proteins and lipids to be
incorporated in the plasma membrane.
Regulated Exocytosis
Here, the vesicle containing secretory proteins releases its contents or membrane
receptors etc. to the outside of the cell or to the plasma membrane on receiving a signal

such as Ca increase or hormone etc.

2+

The regulated exocytosis includes:

(i) The transport of Glut–4 (Glucose Transporter–4) containing vesicles to plasma
membrane of adipose and muscle cells when insulin binds to its receptor in these cells
ii) Secretion of digestive enzymes enclosed in vesicles by acinar cells of pancrease
directly into the lumen of the intestines on receiving hormonal signal.
(iii) Release of acetylcholine from vesicles containing acetyl choline which fuse with

cell membrane at synaptic when Ca level rises as a result of the opening of voltage
2+

gated Ca2+ channels in neurons.

(iv) In the Ca
2+ induced formation of a barrier to further sperm penetration after

fertilization of an ovum.
Insulin stimulation of GLUT–4 transporters of plasma membrane of myocytes by exocytosis of
GLUT–4 membranous vesicles. When insulin is withdrawn, the process is reversed by endocytosis
Exocytosis is thus a general term used to denote vesicle transport and fusion at
plasmamembrane and the release of its contents. It is the final step in the secretory
pathway that begins at endoplasmic reticulum, passes through the Golgi apparatus and
ends outside the cell. Sorting of contents in the vesicles occurs in the endoplasmic
reticulum, golgi or post golgi compartments and are modified enzymatically into their
mature form ready for delivery. Vesicle formation for transport between golgi involves
a mechanism in which GTP–protein complexes bind to the vesicle membrane which
results in binding of COP (for coat protein) molecules and four other proteins to form a
complex called Coatamer around the vesicle. Budding of the vesicle at the binding site
leads to the formation of a coated (non is clathrin coated) vesicle which play a role in
intra golgi transport.
These coated vesicles uncoat at the target membrane as a result of GTP hydrolysis to
GDP by Rab proteins in mammals, which are GTPases. The Rab proteins play a crucial
role where GTP hydrolysis is the signal for uncoating, docking and fusion of vesicles to
its target membrane. Recognition of the vesicle for the target membrane sites is via
special recognition proteins such as:
v–SNARE (where v is for vesicle and SNARE, soluble NSF attachment protein receptor, located
in the external surface of the vesicle). t–SNARE (t is for target vesicle) is the partner protein
located on the cytosolie face of the target membrane which is plasma membrane in case of
exocytosis. Two other proteins, NSF, N–ethyl maleimide sensitive factor) and SNAP (a soluble
NSF attachment Protein) besides other proteins are important in docking and fusion of vesicle
in animals and yeast cells. NSF may act to prime SNAREs before docking and to recycle SNAREs
after fusion. Specific v–and t– SNARE are associated with exocytosis. Different combinations of
SNAREs and other proteins are characteristic of other docking events.
Docking is the process by which the vesicle is fixed beneath the target membrane or
plasma membrane on the cytosolic side before fusion requiring molecular recognition
between vesicle and the target membrane
The fusion of the vesicles at the plasma membrane leads to the formation of fusion pore
which is a channel that passes through the vesicle and the plasma membrane and
allows the content of the vesicle to move out to the extracellualr milieu or compartment.
After the transient fusion pore opening, as the contents of the vesicle are released, there
is a rapid closure of the pore. Alternatively, the pore formation is followed by full pore
opening with incorporation of vesicle membrane into the plasma membrane. Both
transient and permanent fusion can be found in the same animal.
Exocytosis by fusion of vesicles with the plasmamembrane
Endocytosis
Endocytosis is the internalization of material from the extracellular milieu. It is the
process by which a portion of the cell membrane forms a pit or invagination around
large molecules (food particle, protein, virus, bacteria etc.), engulfs or encloses it by
forming a new intracellular membrane enclosed vesicles which is released inside the
cell. Also, membrane proteins, receptors and transporters are endocytosed e.g. GLUT–4,
a glucose transporter in muscle and adipose cells is endocytosed when glucose concentration in
blood falls and insulin dissociates from its receptor
Endocytosis is of 3 types:
(i) Phagocytosis (Cell Eating) when the substance ingested is a solid such as food in
case of protozoa or bacterial cells in case of white blood cells.
(ii) Pinocytosis (cell drinking) when liquid is taken in e.g. human egg cell takes
nutrients from the “nurse cells” by pinocytosis.
(iii) Receptor Mediated endocytosis : In this type material to be taken up, first binds
to a specific protein receptor in the plasma membrane and then the receptor-
material (ligand) complex is engulfed as such in a vesicle – the clathrin coated
vesicle which is endocytosed.
Most cells in the body take up external particles or molecules by receptor mediated
endocytosis and is a widely used pathway for cholesterol, ferritin, cell growth factors,
some viruses etc.
Cell Signaling and Membrane Receptors
Cell Signaling
Cell signaling is the process by which a signal on the surface of the cell is relayed across
its membrane to a specific cellular response inside the cell. This has been conserved
during evolution and developed in prokaryotes and adopted by multicellular
organisms or animals.
The activities of different cells within tissues or organs need to be regulated or
coordinated in a controlled pathway by specific communications. This type of control is
dependent on release of signal molecules from one cell and its recognition and the
delivery of the signals to the target cells. This is possible when the target cell has the
necessary receptor molecules to receive the signals. Such receptor molecules are
allosteric proteins and the signal is referred to as a ligand, whose binding to the
receptor protein is highly specific and induces a conformational change in the receptor
proteins or causes dimerization of the receptor by bringing the cytoplasmic domains
together which triggers the relay of message to affect a change in cellular or biochemical
function or gene expression.
Thus (i) only the cell types which contain the specific receptors for the ligand (signaling
molecules) can respond and cause changes in the cellular function or activities. (ii) The
changes in the metabolic or cellular activity

In some case, binding of different signals or hormones to their specific receptors may
lead to the same response e.g. epinephrine and glucagon binding to their respective
receptors, cause an increase in blood sugar. On the other hand, the same ligand, e.g.
acetyl choline causes different responses by binding to different receptors. It binds to
1. Nicotinic receptor which is an ion channel involved in nerve conduction and
2. Muscarinic receptor which acts through G protein and Phospho lipase C and inositol
triphophate, IP3.

The process by which the message from the ligand or signal molecule is transmitted to
the target cell to modify its activity or gene expression is called signal transduction.
Signal transduction is modular and consists of several components such as:
1. Signaling Ligand (the first messenger).
2. Receptor which binds the signal (ligand) specifically to form receptor – ligand complex
and conveys the message to induce metabolic response.
In some cases, an intermediate component such as G proteins (GTP binding
proteins) relays the message from Receptor-ligand complex to an effector enzyme.
3. In some cases, a second messenger or an activated protein or enzyme carries out the
desired effect on the enzyme activities of a metabolic pathway or may affect gene
expression.
The modular nature of signal transduction allows an amplification of the message at
each successive step and diversity of metabolic responses on the same and different
operating systems.
In each case
(i) The receptor protein is characterized by a binding specificity for a particular
ligand (similar to that of an enzyme for its substrate, but here, no enzyme reaction
occurs and neither the ligand nor the receptor undergo any chemical change) and
(ii) The resulting receptor–ligand complex exhibits effector specificity i.e. mediates a
specific cellular response. The cooperative or allosteric changes in receptor on
binding its specific ligand results in large changes in receptor activation,
(iii) Amplification is by an enzyme cascade which results when an enzyme
associated with the receptor is activated which in turn activates second enzyme and
so on.
In most receptor–ligand systems, the only function of the ligand is to bind to the receptor and
change its properties and provide information for the presence of the specific signal in the
environment. The ligand is neither metabolized to any useful products nor has any enzymatic
activity.
The general principal in cell signaling, therefore, involves ultimately a series of
phosphorylation and dephosphorylation steps of either
• enzymes to convert them from an inactive to an active state on a cascade
pathway or
• (ii) nonenzymic protein to eventually lead to the desired effect.
The importance of phosphorylation and dephosphorylation by kinases / phosphatase
in the control of metabolic pathways or cellular response to hormones was elucidated
by Sutherland for the effect of epinephrine on glycogenolysis at molecular level.

The cellular response to a signal can be terminated when the ligand is removed from its
receptor or the ligand binding receptor is inactivated or by degradation of the second
messenger or the ligand.
Signal Molecules
Some of the signaling molecules are:
(i) Hormones (ii) Neurotransmitters (iii) Pheromones (iv) Nutrients (v) Developmental
Signals (vi) Interleukin (vii) Light (viii) Antigens (ix) Cytokines (x) Nitric Oxide

The signaling molecules can thus include proteins, large and small peptides,
catecholamines, thyroxine, steroids, eicosanoid, vitamin D3, prostaglandin, nitric oxide
etc.
 The main events in membrane receptor mediated signaling

In animals, the extra cellular, secretory signaling molecules can be classified based on
the distance over which the signal acts in to the following types.
(i) Endocrine Signaling: Where the ligand released by the cell affects target cells distant
from their site of synthesis in ductless glands and released directly in the blood
and carried by blood to the target cells e.g. insulin hormone, epinephrine,
glucagon etc.
(ii) Paracrine Signaling, when the signaling molecule released by the cell only affects
target cells which are nearby e.g. neurotransmitters and neurohormones in nerve
conduction and prostaglandin, polypeptide growth hormones.
(iii) Autocrine Signaling, where the cell respond to the signaling molecules they
themselves release e.g. growth factors, T–cell proliferation.
(iv) In addition, some membrane, bound proteins on one cell can directly signal on
adjacent cell by interacting with receptors on adjacent cells.
Beside these intracellular or intercellular signaling, there is another type of signaling,
that by Pheromones. Pheromones are chemicals released by many organisms and are
sexual attractants. They alter the behaviour and gene expression of other organisms of
the same species. Some have other functions in species such as ants that have complex
social interactions. Yeast mating type factors, the small polypeptides, or pheromones,
are well understood pheromone mediated cell-cell signaling. Some algae and animals
also release pheromones into air or water to attract members of the other sex.

Receptors
Receptors for cell signaling are broadly classified as:
(i) Intracellular receptors
(ii) Membrane or Cell Surface Receptors.
Intracellular Receptors
Signaling molecules which are lipophilic such as steroid hormones and the thyroxine,
and nitric oxide (NO) are permeable and can enter the cell and bind to intracellular
receptors. The steroids bind to cytosolic or nuclear protein receptors and modulate
specific gene transcription (usually at the initiation of transcription). Nitric Oxide (NO)
binds to a cytosolic receptor which is a guanylate cyclase and produces cGMP.
Cell Surface Receptors
Signaling molecules or ligands that are hydrophilic such as proteins, amino and
derivatives etc. are impermeable to cell membrane and thus transmit their messages by
binding to cell surface receptors e.g. epinephrine, glucagon, insulin, epidermal growth
factor, neuro transmitters. Prostaglandin and NO also bind to cell surface receptors
which are broadly grouped as:
1. G–Protein Coupled Receptors
2. Ion Channel Receptors
3. Receptors with intrinsic enzyme activity:
Receptor guanylate cyclase; Receptor tyrosine kinases (RTKs); Receptor tyrosine
phosphatase and Receptor serine–threonine kinases
4. Cytokine Receptor Super Family
5. Adhesion protein receptors in the plasma membrane of the cell
These cell adhesion protein receptors are involved in cell–cell recognition and also carry
information between extracellular matrix and cytoskeleton.
Intracellular Second Messengers
Binding of the ligand to some of the cell surface membrane receptors induces formation
of the second messenger whereas ligand binding to others does not.
The intracellular second messengers are soluble, low molecular weight, non-protein,
diffusible molecules. Among the important second messengers are:
(i) 3’,5’– Cyclic AMP (cAMP) (ii) 3’,5’–Cyclic GMP (cGMP) (iii) Inositol–1, 4, 5–
2+
triphosphate (IP3) (iv) 1,2,–diacylglycerol (DAG) (v) Ca .

The elevated concentration of one or more of each of the second messenger triggers a
rapid change in the activity of one or more enzyme(s) or non–enzymic protein(s) which
affect metabolic pathways such as uptake and utilization of glucose by insulin or release of
glucose by epinephrine.
The second messenger can also control proliferation and differentiation of cells,
regulating specific gene transcription.