BT8305 – Cell Biology

UNIT III
TRANSPORT ACROSS CELL MEMBRANE

Passive Transport and Uses of Transport Systems:

Gases, such as O2 and CO2, and small, uncharged polar molecules, such as urea and ethanol, can
readily move by passive (simple) diffusion across an artificial membrane composed of pure
phospholipid or of phospholipid and cholesterol .
Such molecules also can diffuse across cellular membranes without the aid of transport proteins.
No metabolic energy is expended because movement is from a high to a low concentration of the
molecule, down its chemical concentration gradient. Such transport reactions are spontaneous
because they have a positive _Svalue (increase in entropy) and thus a negative _G (decrease in free
energy).
The relative diffusion rate of any substance across a pure phospholipid bilayer is proportional to
its concentration gradient across the layer and to its hydrophobicity and size; charged molecules
are also affected by any electric potential across the membrane. When a phospholipid bilayers
separates two aqueous compartments, membrane permeability can be easily determined by adding
a small amount of radioactive material to one compartment and measuring its rate of appearance
in the other compartment.
The greater the concentration gradient of the substance, the faster its rate of diffusion across a
bilayer.
The hydrophobicity of a substance is measured by its partition coefficient K, the
equilibrium constant for its partition between oil and water. The higher a substance’s partition
coefficient, the more lipid-soluble it is.
The first and rate-limiting step in transport by passive diffusion is movement of a molecule from
the aqueous solution into the hydrophobic interior of the phospholipid bilayer, which resembles
oil in its chemical properties.
This is the reason that the more hydrophobic a molecule is, the faster it diffuses across a pure
phospholipid bilayer.
If a transported substance carries a net charge, its movement is influenced by both its concentration
gradient and the membrane potential, the electric potential (voltage) across the membrane. The
combination of these two forces, called the electrochemical gradient, determines the energetically
favorable direction of transport of a charged molecule across a membrane.
The electric potential that exists across most cellular membranes results from a small imbalance
in the concentration of positively and negatively charged ions on the two sides of the membrane.

Uses
 Movement of virtually all molecules and ions across cellular membranes is mediated by
selective membrane transport proteins embedded in the phospholipid bilayer.
Because different cell types require different mixtures of low-molecular-weight compounds, the
plasma membrane of each cell type contains a specific set of transport proteins that allow only
certain ions and molecules to cross.
Similarly, organelles within the cell often have a different internal environment from that of the
surrounding cytosol, and organelle membranes contain specific transport proteins that maintain
this difference.
Types of transport process:
Two types of transport process occur across the membrane.
1. Non-mediated transport
2. Mediated transport

Non-mediated transport occurs through the simple diffusion process and the driving force for the
transport of a substance through a medium depends on its chemical potential gradient.
Whereasmediated transportrequires specific carrier proteins. Thus, the substance diffuses in the
direction that eliminates its concentration gradient; at a rate proportional to the magnitude of this
gradient and also depends on its solubility in the membrane’s non-polar core. Mediated transport
is classified into two categories depending on the thermodynamics of the system:
1. Passive-mediated transport, or facilitated diffusion:In this typeof process a specific molecule
flows from high concentration to low concentration.
2. Active transport:In this typeof process a specific molecule is transported from low concentration
to high concentration, that is, against its concentration gradient. Such an endergonic process must
be coupled to a sufficiently exergonic process to make it favorable (ΔG <0).
Figure 2: Mediated transport. (A) Passive transport and (B) Active transportNPTEL –
Biotechnology – Cell Biology Joint initiative of IITs and IISc – Funded by MHRD Page 5 of 120
Passive mediated transport:
Substances that are too large or polar diffuse across the lipid bilayer on their own through
membrane proteins called carriers, permeases, channels and transporters. Unlike active transport,
this process does not involve chemical energy. So the passive mediated transport is totally
dependent upon the permeability nature of cell membrane, which in turn, is function of
organization and characteristics of membrane lipids and proteins .
Types of passive transport:

1. Diffusion:
The process of the net movement of solutes from a region of high concentration to a region of low
concentration is known as diffusion. The differences of concentration between the two regions are
termed as concentration gradient and the diffusion continues till the gradient has been vanished.
Diffusion occurs down the concentration gradient.
Figure 3: Diffusion.Extracellular space contains high concentration of solutes than intracellular
space and hence the solutes move from extracellular space to intracellular space till there is no
concentration gradient between the spaces.

2. Facilitated diffusion :
The process of the movement of molecules across the cell membrane via special transport proteins
that are embedded within the cellular membrane is known as facilitated diffusion or called carrier-
mediated diffusion. Many large molecules, such as glucose, are insoluble in lipids and too large to
fit into the porins, therefore, it will bind with its specific carrier proteins, and the complex will
then be bonded to a receptor site and moved through the cellular membrane.

Figure 4: Facilitated transport.Movement of the solutes from extracellular space to intracellular

space via carrier proteins and down its concentration gradient.

3. Filtration:

Filtration is the process of the

movement of water and solute
molecules across the cell
membrane due to hydrostatic
pressure generated by the
system. Depending on the size
of the membrane pores, only
solutes of a certain size may
pass through it. The membrane
pores of the Bowman's capsule
in the kidneys are very small,
and only albumins (smallest of
the proteins) can filter through.
On the other hand, the
membrane pores of liver cells
are extremely large, to allow a
variety of solutes to pass through and be metabolized.

Figure 5: Filtration
4. Osmosis:
Osmosis is the type of diffusion of water molecules across a semi- permeable membrane, from a
solution of high water potential to a region of low water potential. A cell with a less negative water
potential will draw in water but this depends on other factors as well such as solute potential
(pressure in the cell e.g. solute molecules) and pressure potential (external pressure e.g. cell wall).

Figure 6: Osmosis.(A) In hypertonic solution, there are more solute molecules outside the cell,
which causes the water to be sucked in that direction which leads to the shrinkage of cells. (B) In
isotonic solution, there is equal concentration of solute on both sides, henceforth the water with
move back in forth. (C) In hypotonic solution, there are less solute molecules outside the cell, since
salt sucks and water will move inside the cell. The cell will gain water and grow larger, and finally
burst.

Active Transport

Active transport: moving against a gradient

To move substances against a concentration or electrochemical gradient, a cell must use energy.
Active transport mechanisms do just this, expending energy (often in the form of ATP) to maintain
the right concentrations of ions and molecules in living cells. In fact, cells spend much of the
energy they harvest in metabolism to keep their active transport processes running. For instance,
most of a red blood cell’s energy is used to maintain internal sodium and potassium levels that
differ from those of the surrounding environment.

Active transport mechanisms can be divided into two categories. Primary active
transport directly uses a source of chemical energy (e.g., ATP) to move molecules across a
membrane against their gradient. Secondary active transport (cotransport), on the other hand,
uses an electrochemical gradient – generated by active transport – as an energy source to move
molecules against their gradient, and thus does not directly require a chemical source of energy
such as ATP. We’ll look at each type of active transport in greater detail below.

Primary active transport

One of the most important pumps in animal cells is the sodium-potassium pump, which moves
Na^++start superscript, plus, end superscript out of cells, and K^++start superscript, plus, end
superscript into them. Because the transport process uses ATP as an energy source, it is considered
an example of primary active transport.
Not only does the sodium-potassium pump maintain correct concentrations of Na^++start
superscript, plus, end superscript and K^++start superscript, plus, end superscript in living cells,
but it also plays a major role in generating the voltage across the cell membrane in animal cells.
Pumps like this, which are involved in the establishment and maintenance of membrane voltages,
are known as electrogenic pumps. The primary electrogenic pump in plants is one that pumps
hydrogen ions (H^++start superscript, plus, end superscript) rather than sodium and
potassium^{2,3}2,3start superscript, 2, comma, 3, end superscript.
The sodium-potassium pump cycle

Figure showing the transport cycle of the sodium-potassium pump.

The sodium-potassium pump transports sodium out of and potassium into the cell in a repeating
cycle of conformational (shape) changes. In each cycle, three sodium ions exit the cell, while two
potassium ions enter. This process takes place in the following steps:
To begin, the pump is open to the inside of the cell. In this form, the pump really likes to bind (has
a high affinity for) sodium ions, and will take up three of them.
When the sodium ions bind, they trigger the pump to hydrolyze (break down) ATP. One phosphate
group from ATP is attached to the pump, which is then said to be phosphorylated. ADP is released
as a by-product.
Phosphorylation makes the pump change shape, re-orienting itself so it opens towards the
extracellular space. In this conformation, the pump no longer likes to bind to sodium ions (has a
low affinity for them), so the three sodium ions are released outside the cell.
In its outward-facing form, the pump switches allegiances and now really likes to bind to (has a
high affinity for) potassium ions. It will bind two of them, and this triggers removal of the
phosphate group attached to the pump in step 2.
With the phosphate group gone, the pump will change back to its original form, opening towards
the interior of the cell.
In its inward-facing shape, the pump loses its interest in (has a low affinity for) potassium ions, so
the two potassium ions will be released into the cytoplasm. The pump is now back to where it was
in step 1, and the cycle can begin again.
This may seem like a complicated cycle, but it just involves the protein going back and forth
between two forms: an inward-facing form with high affinity for sodium (and low affinity for
potassium) and an outward-facing form with high affinity for potassium (and low affinity for
sodium). The protein can be toggled back and forth between these forms by the addition or removal
of a phosphate group, which is in turn controlled by the binding of the ions to be transported.
How the sodium-potassium pump generates a membrane potential
How, exactly, does the sodium-potassium pump establish a voltage across the membrane? It’s
tempting to simply make an argument based on stoichiometry: for every three ions of sodium that
move out, only two ions of potassium move in, resulting in a more negative cell interior. While
this charge ratio does make the cell’s interior slightly more negative, it actually accounts for only
a tiny fraction of the sodium-potassium pump’s effect on membrane potential.
Instead, the sodium-potassium pump acts primarily by building up a high concentration of
potassium ions inside the cell, which makes potassium’s concentration gradient very steep. The
gradient is steep enough that potassium ions will move out of the cell (via channels), despite a
growing negative charge on the interior. This process continues until the voltage across the
membrane is large enough to counterbalance potassium’s concentration gradient. At this balance
point, the inside of the membrane is negative relative to the outside. This voltage will be
maintained as long as K^++start superscript, plus, end superscriptconcentration in the cell stays
high, but will disappear if K^++start superscript, plus, end superscript stops being
imported^{4,5}4,5start superscript, 4, comma, 5, end superscript.
For more explanation of how the voltage across the membrane is established, take a look at
the membrane potential article in the neurobiology section.
Secondary active transport
The electrochemical gradients set up by primary active transport store energy, which can be
released as the ions move back down their gradients. Secondary active transport uses the energy
stored in these gradients to move other substances against their own gradients.
As an example, let's suppose we have a high concentration of sodium ions in the extracellular
space (thanks to the hard work of the sodium-potassium pump). If a route such as a channel or
carrier protein is open, sodium ions will move down their concentration gradient and return to the
interior of the cell.
In secondary active transport, the movement of the sodium ions down their gradient is coupled to
the uphill transport of other substances by a shared carrier protein (a cotransporter). For instance,
in the figure below, a carrier protein lets sodium ions move down their gradient, but simultaneously
brings a glucose molecule up its gradient and into the cell. The carrier protein uses the energy of
the sodium gradient to drive the transport of glucose molecules.

Diagram of a sodium-glucose cotransporter, which uses the energy stored in a sodium ion gradient
to transport glucose "uphill" against its gradient. The cotransporter accomplishes this by physically
coupling the transport of glucose to the movement of sodium ions down their concentration
gradient.
In secondary active transport, the two molecules being transported may move either in the
same direction (i.e., both into the cell), or in opposite directions (i.e., one into and one out of the
cell). When they move in the same direction, the protein that transports them is called a symporter,
while if they move in opposite directions, the protein is called an antiporter.
Membrane transport is assisted by various facilitators to ease their job. We will study a few of
them in detail.
Permeases
Permeases are a class of membrane transport proteins which facilitate the diffusion of a specific
molecule by passive mediated transport. These are divided into following types:
1. Lactose permease: It is a transmembrane protein that consists of N- and C- terminal domains,
each consisting of six membrane-spanning alpha helices in a symmetrical fashion. These two
domains are well separated and are joined by a single stretch of polypeptide. There are six side
chain amino acids that play an important role in the active transport of lactose through the protein.
Glutamic Acid 126 , Arginine 144 , and Glutamic Acid 269 plays role in substrate binding activities
where as Arginine 302 , Histidine 322 , and Glutamic Acid 325 plays a significant role in proton
translocation throughout the transport process. These side chains, make up the active site of the
protein and found within the large internal hydrophilic cavity of the lactose permease where the
substrate is received for transport and it is the location from which it is sent into the cell.
It is an active co-transport that facilitates the passage of lactose across the phospholipid bi-
layer of the cell membrane by using the inwardly directed H+ electrochemical gradient as its
driving force. The proton gradient is metabolically generated through oxidative metabolism. The
electrochemical potential gradient created by both these systems is used mainly to drive the
synthesis of ATP. As a result, the lactose is accompanied from the periplasam to the cytoplasm of
the cell by an H+ proton.
Lactose permease has two major conformational states:
1. E-1, which has a low-affinity lactose-binding site facing the interior of the cell.
2. E-2, which has a high-affinity lactose-binding site facing the exterior of the cell.
Figure 1: Schematic diagram for the cotransport of H+ and lactose by lactose permease in E.Coli.
H+ binds first to E-2 outside the cell, followed by lactose. They are sequentially released from E-
1 inside the cell. E-2 must bind to lactose and H + in order to change the conformation to E-1,
thereby cotransporting these substances in the cell. E-1 changes the conformation to E-2 when
neither lactose nor H+ is bound, thus completing the transport cycle.
Lactose permease is a membrane protein which is a
member of the major facilitator superfamily.
Lactose permease can be classified as a symporter,
which uses the proton gradient towards the cell to
transport β-galactosides such as lactose in the same
direction into the cell.
The protein has twelve transmembrane helices and
exhibits an internal two-fold symmetry, relating the
N-terminal six helices onto the C-terminal helices. It
is encoded by the lacY gene in the lac operon.
The sugar lies in a pocket in the center of the protein
which is accessible from the periplasm. On binding,
a large conformational change takes place which makes the sugar binding site accessible from the
cytoplasm.
Mechanism: hydrogen from the outside of the cell binds to a carboxyl group on the enzyme that
allows it to undergo a conformational change. This form of lactose permease can bind lactose from
outside the cell. The enzyme then everts and lactose is transported inward.
2.β-galactoside permease is
a membrane-bound transport
protein that facilitates the
uptake of β-galactosides
across the cell. The common
example is melibiose carrier
protein from Klebsiella
pneumonia, which is capable
of using hydrogen and
lithium cations as coupling
cations for cotransport,
depending on the particular sugar transported (H+-melibiose, Li+-lactose).
3. Amino acid permeases are integral
membrane proteins involved in the
transport of amino acids into the cell.
One of the examples of amino acid
permease is histidine permease which is
a bacterial ABC protein in E.coli and
located in the periplasmic space of cell.
Histidine binding protein binds histidine
tightly and directs it to T sub-units of
permease, through which hisitidine
crosses the plasma membrane along
with ATP hydrolysis.
1 - A si transport through the histidine permease requires the interaction of the closed, liganded
form of HisJ with the HisQMP2 complex. Once HisJ binds its substrate, L-histidine in this case,
it shuttles its cargo
to the

HisQMP2 complex.
2 - The interaction of HisJ with the HisQMP2 complex induces a conformational change within
the complex. It is believed that the signal is primarily transduced from HisJ to the HisQ
subunit. There is some evidence that HisM also plays a role in the signal transduction, but its role
is minor at best. The induced conformational change within the complex stimulates the two HisP
subunits to hydrolyze the bound ATP. This reaction provides the energy necessary to translocate
the substrate across the inner membrane.
3 - Once ATP hydrolysis occurs, the substrate is translocated from the periplasm into the cytosol
of the bacteria.
4 - In order for the next cycle of transport to occur, ADP dissociates from the HisP subunits while
HisJ dissociates from the transmembrane subunits.
5 - ATP is loaded onto the HisP subunits and the complex is ready for the next cycle of transport.

Ion channels

Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore.
Their functions include establishing a resting membrane potential, shaping action potentials and
other electrical signals by gating the flow of ions across the cell membrane, controlling the flow
of ions across secretory and epithelial cells, and regulating cell volume. Ion channels are present
in the membranes of all excitable cells. Ion channels are one of the two classes of ionophoric
proteins, the other being ion transporters.
Their classification as molecules is referred to as channelomics.
Classification
There are over 300 types of ion channels just in the cells of the inner ear. Ion channels may
be classified by the nature of their gating, the species of ions passing through those gates, the
number of gates (pores) and localization of proteins.
Further heterogeneity of ion channels arises when channels with different constitutive subunits
give rise to a specific kind of current. Absence or mutation of one or more of the contributing types
of channel subunits can result in loss of function and, potentially, underlie neurologic diseases.
Classification by gating
Ion channels may be classified by gating, i.e. what opens and closes the channels. For example,
voltage-gated ion channels open or close depending on the voltage gradient across the plasma
membrane, while ligand-gated ion channels open or close depending on binding of ligands to the
channel.
Voltage-gated
Voltage-gated ion channels
open and close in response to
membrane potential.
Voltage-gated sodium
channels: This family contains
at least 9 members and is
largely responsible for action
potential creation and
propagation. The pore-forming
α subunits are very large (up to
4,000 amino acids) and consist
of four homologous repeat
domains (I-IV) each comprising six transmembrane segments (S1-S6) for a total of 24
transmembrane segments. The members of this family also coassemble with auxiliary β subunits,
each spanning the membrane once. Both α and β subunits are extensively glycosylated.

Voltage-gated calcium
channels: This family
contains 10 members,
though these members
are known to coassemble
with α2δ, β, and γ
subunits. These channels
play an important role in
both linking muscle
excitation with
contraction as well as
neuronal excitation with
transmitter release. The α subunits have an
overall structural resemblance to those of
the sodium channels and are equally large.

Cation channels of sperm: This small

family of channels, normally referred to as
Catsper channels, is related to the two-
pore channels and distantly related to TRP
channels.
 Voltage-gated potassium channels (KV): This family
contains almost 40 members, which are further
divided into 12 subfamilies. These channels are
known mainly for their role in repolarizing the cell
membrane following action potentials. The α
subunits have six transmembrane segments,
homologous to a single domain of the sodium
channels. Correspondingly, they assemble as
tetramers to produce a functioning channel.

Voltage-gated proton channels: Voltage-gated proton channels open with depolarization, but in a
strongly pH-sensitive manner. The result is that these channels open only when the electrochemical
gradient is outward, such that their opening will only allow protons to leave cells. Their function
thus appears to be acid extrusion from cells. Another important function occurs in phagocytes (e.g.
eosinophils, neutrophils, macrophages) during the "respiratory burst." When bacteria or other
microbes are engulfed by phagocytes, the enzyme NADPH oxidase assembles in the membrane
and begins to produce reactive oxygen species (ROS) that help kill bacteria. NADPH oxidase is
electrogenic, moving electrons across the membrane, and proton channels open to allow proton
flux to balance the electron movement electrically.

Ligand-gated (neurotransmitter)
Also known as
ionotropic receptors, this
group of channels open
in response to specific
ligand molecules
binding to the
extracellular domain of
the receptor protein.
Ligand binding causes a
conformational change
in the structure of the
channel protein that
ultimately leads to the
opening of the channel
gate and subsequent ion
flux across the plasma
membrane. Examples of
such channels include
the cation-permeable
"nicotinic"
Acetylcholine receptor, ionotropic glutamate-gated receptors, acid sensing ion channels
(ASICs),[10] ATP-gated P2X receptors, and the anion-permeable γ-aminobutyric acid-gated
GABAA receptor.
Ion channels activated by second messengers may also be categorized in this group, although
ligands and second messengers are otherwise distinguished from each other.

Ligand-gated ion channels are large,

multisubunit (4 or 5 subunits)
receptors that form a membrane ion
channel that, when open, allows the
passage of Na+, K+, Ca++, or Cl−. Once
the receptor/channel complex is
activated, the membrane potential may
become depolarized or hyperpolarized,
depending on the direction of the ion
flow and the ion involved. Typically,
each subunit contains 4 hydrophobic
transmembrane domains linked by
hydrophilic groups. Activation of these
channels leads to a rapid (msec)
response, allowing ions to flow down
their electrochemical
gradients. Nicotinic
cholinergic, GABA-A, and the 5-
hydroxytryptamine3 (5HT3) receptorare
examples of ligand-gated ion channel sites.

ACETYL CHOLINE RECEPTOR:

The acetyl choline receptor is ligand gated ion channel. It is essential in the passage of an
electriacal signal from a motor neuron to a muscle fiber at the neuromuscular junction. Acetyl
choline released by motor neuron diffuses a few micro meters to the plasma membrane of a
myocytes which binds to the acetyl choline receptor. This bring conformational changes to the
acetyl choline receptor causing the ion channel to open. Resulting inward movement of positive
charge depolarize the plasma membrane of the myocyte triggering contraction. Acetyl choline
receptor allows only sodium, calcium and potassium. acetyl choline receptor are made up of 5
subunits two α and one , and . Two alpha are acetyl choline binding site. All this subunits are
made up of tertiary structure. acetyl choline receptors are intergral proteins which protrude both in
cytosolic and extracellular surface. But it narrows as it passes throught the lipid bilayer. In center
of lipid bilayer channel is closed by hydrophobic leucine side chain from the M2 helices.

The nicotinic acetylcholine receptor is an example of a ligand-gated ion channel. It is composed

of five subunits arranged symmetrically around a central conducting pore. Upon binding
acetylcholine, the channel opens and allows diffusion of sodium (Na +) and potassium (K+) ions
through the conducting pore.
ATP Pumps

ATPases (EC 3.6.1.3, adenylpyrophosphatase, ATP monophosphatase, triphosphatase, SV40 T-

antigen, adenosine 5'-triphosphatase, ATP hydrolase, complex V (mitochondrial electron
transport), (Ca2+ + Mg2+)-ATPase, HCO3−-ATPase, adenosine triphosphatase) are a class
of enzymes that catalyze the decomposition of ATP into ADP and a free phosphate ion or the
inverse reaction. This dephosphorylation reaction releases energy, which the enzyme (in most
cases) harnesses to drive other chemical reactions that would not otherwise occur. This process is
widely used in all known forms of life.
Some such enzymes are integral membrane proteins (anchored within biological membranes), and
move solutes across the membrane, typically against their concentration gradient. These are
called transmembrane ATPases.

There are different types of ATPases, which can differ in function (ATP synthesis and/or
hydrolysis), structure (F-, V- and A-ATPases contain rotary motors) and in the type of ions they
transport.

F-ATPases (F1FO-ATPases) in mitochondria, chloroplasts and bacterial plasma membranes are

the prime producers of ATP, using the proton gradient generated by oxidative
phosphorylation (mitochondria) or photosynthesis (chloroplasts).
V-ATPases (V1VO-ATPases) are primarily found in eukaryotic vacuoles, catalysing ATP
hydrolysis to transport solutes and lower pH in organelles like proton pump of lysosome.
A-ATPases (A1AO-ATPases) are found in Archaea and function like F-ATPases
P-ATPases (E1E2-ATPases) are found in bacteria, fungi and in eukaryotic plasma membranes and
organelles, and function to transport a variety of different ions across membranes.
E-ATPases are cell-surface enzymes that hydrolyze a range of NTPs, including extracellular ATP.

P-ATPase
P-ATPases (sometime known as E1-E2 ATPases) are found in bacteria and also in eukaryotic
plasma membranes and organelles. Its name is due to short time attachment of inorganic phosphate
at the aspartate residues at the time of activation. Function of P-ATPase is to transport a variety of
different compounds, like ions and phospholipids, across a membrane using ATP hydrolysis for
energy. There are many different classes of P-ATPases, which transports a specific type of ion. P-
ATPases may be composed of one or two polypeptides, and can usually take two main
conformations, E1 and E2.

ATPase pumps
ATPase pumps use the energy from ATP to transport ions against their concentration gradients.
A lot of energy in the cell (25% of the ATP) is used up by the ATPase pumps. Used for many
different ions. Essential to maintain the Na+, K+ and Ca+2 concentration gradients that we will
be talking about when we discuss cotransport, action potentials, and muscle contraction.
 We will discuss mostly P-class pumps a class of ATPase pump that is phosphorylated to drive
transport. There are a number of other classes diagrammed above and listed in Table 15-2 in
your text book.

P-class pumps
Na+/K+ ATPase pump
P-class ion pumps:
These are multipass transmembrane proteins having two identical catalytic α-subunits that
contain an ATP binding site. Some have two smaller β-subunits that usually have regulatory
functions. During the transport process or pumping cycle at least one of the α- subunit must be
phosphorylated and the H+ ions are thought to move through the phosphorylated subunit. This
class includes many ion pumps that are responsible for setting up and maintaining gradients of
Na+, K+, H+ and Ca2+ across the cell membrane.
The common P-type pump is mostly found in parietal cells of the mammalian stomach
which transport protons (H+ ions) out of cell and K+ ions into the cell and is mainly responsible
for the acidification of the stomach contents. The pump is known as H +/K+ ATPase. It is a
heterodimeric protein. The H+/K+ ATPase transports one H+ from the cytoplasm of the parietal
cell in exchange for one K+ retrieved from the gastric lumen. As an ion pump the H +/K+ ATPase
is able to transport ions against a concentration gradient using energy derived from the hydrolysis
of ATP. Like all P-type ATPases, a phosphate group is transferred from ATP to the H+/K+ ATPase
during the transport cycle.
Another example of P-type pump is Na+/K+ ATPase in the plasma membrane, which generates
low cytosolic Na+ and high cytosolic K+ concentration which is typical of animal cells (discussed
in earlier lecture).
Certain Ca2+ ATPase pump Ca2+ ions out of the cytosol into the external medium while others
pump Ca2+ from the cytosol into the endoplasmic recticulum or into the specialized sarcoplasmic
reticulum, which is more common in muscle cells (discussed in earlier lecture).
F-class ion pumps:
The F class ion pumps contain different transmembrane and cytosolic subunits. They are known
for only transport of protons, in a process that does not involve phosphoprotein intermediate. They
generally behave as reverse proton pump by synthesizing ATP from ADP and Pi by movement of
protons from the exoplasmic to the cytosolic face of the membrane down the proton
electrochemical gradient. Therefore, these pumps are also known as ATP synthases or F 0F1
complex. F-class ion pump is most common in bacteria, yeast and animal mitochondria and also i
n chloroplast.

F0F1 complex is a multi-protein having two components F0 and F1. Both are multimeric proteins.
The F0 component contains three integral membrane proteins named a, b and c. The a and two b
subunits are linked tightly but not to the donut- shaped ring of c subunits. And the F1 component
is water soluble complex of five distinct polypeptides with the composition α3β3γδε. The lower
part of the F1 γ subunit is a coil which fits into the centre of the c-subunit ring of F0 and appears
rigidly attached to it. The F1 ε subunit is rigidly attached to γ and also forms rigid contacts with c
subunits. The F1 and subunits associate in alternating order to form a hexamer αβαβαβ. The F1 δ
subunit is permanently linked to one of the F1 subunits and also to the b subunit of F0. Thus the F0
a and b subunits and the δ subunit and (αβ)3 hexamer of the F1 complex form a rigid structure
anchored in the membrane. The rodlike b subunits form a stator that prevents the (αβ)3 hexamer
from moving while it rests on the γ subunit.

Figure 2: Model of the structure and function of ATP synthase (the F0F1 complex) in the bacterial
plasma membrane.
The F0 portion is built of three integral membrane proteins: one copy of a, two copies of b, and on
average 10 copies of c arranged in a ring in the plane of the membrane. Two proton half-channels
lie at the interface between the a subunit and the c ring. Half- channel I allows protons to move
one at a time from the exoplasmic medium and bind to aspartate-61 in the center of a c subunit
near the middle of the membrane. Half-channel II (after rotation of the c ring) permits protons to
dissociate from the aspartate and move into the cytosolic medium.
0

pumps 2 K+ in and 3 Na+ out

- important for many cellular functions (osmotic balance of cells)
- 3 Na+ out for every 2 K+ ions in
- uses ATP as energy source
- binding of phosphate from ATP drives conformation change that allows ions to be transported to
appropriate sides
- an asparate residue becomes phosphorylated and the energy transfer changes the proteins
conformational shape
- Na+ binding sites switch from high affinity on inside to low affinity on outside to allow for
binding of Na+ on inside and release of Na+ ions on outside.
- K+ binding sites switch from high affinity on outside to low affinity on inside for the same reason
- can be blocked with poisons like ouabain or digitalis
- blocked pump can't transport ions across membrane and the chemical gradients for Na and K
slowly disappear due to continuous action of the leak channels, resting potential removed.
- the potential built up in the Na+ ions will be used by many different processes i.e. cotransporters,
neuronal signaling etc.

Ca+2 ATPase pump

- pumps 2 Ca+2 ions out for every 1 ATP molecule used
- again uses ATP to drive Ca+2 out against a very large concentration gradient
- internal Ca+2 binding sites have a very high affinity (in order to overcome extremely low Ca+2
concentrations inside cell)
- energy transfer from ATP to the aspartate of the Ca+ ATPase causes a protein conformational
change and Ca+2 transported across membrane
- Ca+2 binding sites on outside are low affinity and Ca+2 is released
- the transfer of eneragy from the ATP to the pump triggers a conformational change that moves
the protein and allows the translocation of Ca+2 across the membrane. At the same time the
Ca+2 binding sites change from high to low affinity.
- in muscle cells the Ca+2 ATPase is the major protein found in the membrane of the
sacrcoplasmic reticulum (SR). As we will see the SR is a storage site for Ca+2 that is release to
drive muscle contraction. The Ca+2 ATPase will remove excess Ca+2 from the cytoplasm and
pump it into the lumen of the SR.
80% of the protein in the SR is the Ca+2 ATPase. Therefore this made it easy to purify and study
in liposomes as we discussed in class.

V-class pumps
These pumps transport H+ only.
- found in lysosomes, endosomes and plant vacuoles.
- transport H+ ions to make the lumen or inside of the lysosome acidic (pH 4.5 - 5.0)
- many of these pumps are paired with Cl- channels to offset the electrical gradient that is produced
by pumping H+ across the membrane.

V-class ion pumps:

It is almost similar to F-class ion pumps in structure and function. But
none of their subunits are related to each other. F-class pumps operate
in reverse direction to V-class. These pumps generally function to
maintain low pH of plant vacuoles and lysosome and other acidic
vesicles in animal cells by pumping protons from cytosolic to
exoplasmic face (inside) of membrane against the proton
electrochemical gradient. The acidification between the lysosomal
lumen and cytosol lumen can be maintained by production of ATP by
cells.
These V-class proton pumps contain two domains: a cytosolic
hydrophilic domain (V1) and a transmembrane domain (V0) with
multiple subunits in each domain. Binding and hydrolysis of ATP by
the B subunits in V1 provide the energy for pumping of H+ ions through
the proton-conducting channel formed by the c and a subunits in V0. These V- class proton pumps
are not phosphorylated and dephosphorylated during proton transport.

Figure 3: V-class proton pump

These protons cannot acidify by themselves because a net movement of electric charge occurs.
Only a few protons build up positive H+ ions on exoplasmic face (inside) and for each H+ pumped
across, a negative ion will be left behind on cytosolic face, building negatively charged ions. These
oppositely charged ions attract each other on opposite faces of the membrane, generating a charge
separation, or electric potential, across the membrane. If more protons pumped, the excess positive
ions on exoplasmic face repels other H+ ions and prevents pumping of extra proton long before a
significant transmembrane H+ concentration gradient had been established. If the organelle lumen
or

As H+ is transported into the lysosome Cl- flows in to keep a balance. If Cl- doesn't flow in then
there is rapid build up of
potential (charge) across the
membrane which would block
the further transport of H+.
This would occur long before
the lumen becomes acidic
because not that many ions
need to be transported to
produce the voltage potential.
Other pumps like the H+/K+
ATPase pump (a P-class
pump) transport H+ out and at
the same time K+ in to make
sure there is no voltage
potential created.

ABC (ATP binding

cassettes) superfamily:
The final class of ATP-
powered pumps is a large
family of multiple
membranes. This class
includes several hundred
different transport proteins
found in all organisms ranging
from bacteria to mammals. Each ABC protein is specific for single substrate or group of related
substrate, which may be ions, sugars, amino acids, phospholipids, cholesterol, peptides,
polysaccharides or proteins.

Structure :
All ABC transport protein share a structural organization consisting of four core domains: two
transmembarne (T) domains, forming the passageway through which transported molecules cross
the membrane and two cytosolic ATP-binding (A) domains. The core domains are generally
present in separate polypeptides which are more common in bacterial cell. In others, the core
domains are fused into one or two multidomain polypeptides. ATP binding leads to dimerization
of two ATP-binding domains and ATP hydrolysis leads to their dissociation. Theses structural
changes in the cytosolic domains are thought to be transmitted to the transmembrane segments,
driving cycles of conformational changes that alternately expose substrate-binding sites on one
side of the membrane and then on the other. In this way, ABC transporters use ABC binding and
hydrolysis to transport small molecules across the bilayer.

Some common example of ABC transporters are found in bacterial plasma membranes
which contain amino acid, sugar and peptide transporters. These cells use H + gradient across the
membrane to pump variety of nutrients into the cell. It is also present is mammalian plasma
membrane that contains transporters of phospholipids, small lipophillic drugs, cholesterol and
other small molecules. One example of eukaryotic ABC transporters is multidrug resistance
 (MDR) protein which has the ability to pump
hydrophobic drugs out of the cytosol.
Overexpression of these MDR protein in human
cancer cells, make the cells resistant to variety of
chemically unrelated cytototoxic drugs

ATP-binding cassette transporters (ABC-

transporters) are members of a protein
superfamily that is one of the largest and most
ancient families with representatives in
all extant phyla from prokaryotes to humans.
ABC transporters are transmembrane proteins
that utilize the energy of adenosine
triphosphate (ATP) hydrolysis to carry out
certain biological processes including
translocation of various substrates across
membranes and non-transport-related processes
such as translation of RNA and DNA repair.
They transport a wide variety of substrates across extra- and intracellular membranes,
including metabolic products, lipids and sterols, and drugs. Proteins are classified as ABC
transporters based on the sequence and organization of their ATP-binding cassette (ABC)
domain(s).
ABC transporters are involved in tumor resistance,qq cystic fibrosis and a range of other inherited
human diseases along with both bacterial (prokaryotic) and eukaryotic(including human)
development of resistance to multiple drugs. Bacterial ABC transporters are essential in cell
viability, virulence, and pathogenicity.
ABC transporters are divided into three main functional categories. In prokaryotes, importers
mediate the uptake of nutrients into the cell. The substrates that can be transported include ions,
amino acids, peptides, sugars, and other molecules that are mostly hydrophilic. The membrane-
spanning region of the ABC transporter protects hydrophilic substrates from the lipids of the
membrane bilayer thus providing a pathway across the cell membrane . In gram-negative bacteria,
exporters transport lipids and some polysaccharides from the cytoplasm to the
periplasm. Eukaryotes do not possess any importers. Exporters or effluxers, which are both present
in prokaryotes and eukaryotes, function as pumps that extrude toxins and drugs out of the cell. The
third subgroup of ABC proteins do not function as transporters, but rather are involved in
translation and DNA repair processes.
In bacterial efflux systems, certain substances that need to be extruded from the cell include
surface components of the bacterial cell (e.g. capsular polysaccharides, lipopolysaccharides, and
teichoic acid), proteins involved in bacterial pathogenesis (e.g. hemolysis, heme-binding protein,
and alkaline protease), heme, hydrolytic enzymes, S-layer proteins, competence factors,
toxins, antibiotics, bacteriocins, peptide antibiotics, drugs and siderophores. They also play
important roles in biosynthetic pathways, including extracellular polysaccharide biosynthesis and
cytochrome biogenesis.
 Symporter
Cotransporters use the energy
stored in the electrochemical
gradient of Na+ or H+ ions to power
the uphill movement of another
substance, which may be a small
organic molecule or a different ion.
When the transported molecule
and cotransported ion move in the
same direction, the process is called
symport; when they move in
opposite directions, the process is
called antiport
Most body cells import glucose
from the blood down its
concentration gradient, utilizing one
or another GLUT protein to facilitate
this transport.
However, certain cells, such as
those lining the small intestine and
the kidney tubules, need to import
glucose from the intestinal lumen
against a very large concentration
gradient.
 Such cells utilize a two-
Na+/one-glucose symporter, a
protein that couples import of
one glucose molecule to the
import of two Na+ ions
The two-Na+/glucose
symporter is thought to contain
14 transmembrane _ helices with
both its N- and C-termini
extending into the cytosol. A
truncated recombinant protein
consisting of only the five C-
terminal transmembrane _
helices can transport glucose
independently of Na+ across the
plasma membrane, down its
concentration gradient. This
portion of the molecule thus
functions as a glucose uniporter.
The N-terminal portion of the
protein, including helices 1–9, is required to couple Na+ binding and influx to the transport of
glucose against a concentration gradient.

Na /Ca2+ antiporter
In cardiac muscle cells a three-Na+/one-Ca2+ antiporter, rather than the plasma membrane
Ca2+ ATPase , plays the principal role in maintaining a low concentration of Ca2+ in the cytosol.
The transport reaction mediated by this cationantiportercan be written
3 Na+out + Ca2+ in GIVES 3Na+in +Ca2+ out
Note that the movement of three Na+ ions is required to power the export of one Ca2+ ion from
the cytosol with a [Ca2+] of ≈2 _ 10_7 M to the extracellular medium with a [Ca2+] of 2 _ 10_3
M, a gradient of some 10,000-fold.
As in other muscle cells, a rise in the cytosolic Ca2+ concentration in cardiac muscle triggers
contraction. By lowering cytosolic Ca2+, operation of the Na+/Ca2+ antiporter reduces the
strength of heart muscle contraction.

Cl- /HCO3- antiporter

To cope with the excess OH- ions associated with elevated pH, many animal cells

utilize an anion antiporterthat catalyzes the one-for-one exchange of HCO3-and Cl- across the
plasma membrane.
At high pH, this Cl-/HCO3-antiporter exports HCO3- (which can be viewed as a “complex” of
OH- and CO2) in exchange for Cl- thus lowering the cytosolic pH.
The import of Cl- down its concentration gradient (Cl- medium Cl- cytosol) powers the reaction.
The activity of all three of these antiport proteins depends ,on pH, providing cells with a fine-
tuned mechanism

AGONISTS

An agonist is a chemical that binds to a receptor and activates the receptor to produce a biological
response. Whereas an agonist causes an action, an antagonist blocks the action of the agonist and
an inverse agonist causes an action opposite to that of the agonist.
Types of agonists
Receptors can be activated by either endogenous (such as hormones and neurotransmitters) or
exogenous (such as drugs) agonists, resulting in a biological response. A physiological agonist is
a substance that creates the same bodily responses but does not bind to the same receptor.
An endogenous agonist for a particular receptor is a compound naturally produced by the body
that binds to and activates that receptor. For example, the endogenous agonist for serotonin
receptors is serotonin, and the endogenous agonist for dopamine receptors is dopamine.

A superagonist is a compound that is

capable of producing a greater
maximal response than the
endogenous agonist for the target
receptor, and thus has an efficacy of
more than 100%. This does not
necessarily mean that it is more potent
than the endogenous agonist, but is
rather a comparison of the maximum
possible response that can be
produced inside the cell following
receptor binding.
Full agonists bind (have affinity for)
and activate a receptor, producing full
efficacy at that receptor. One example
of a drug that acts as a full agonist is isoproterenol, which mimics the action of adrenaline at β
adrenoreceptors. Another example is morphine, which mimics the actions of endorphins at μ-
opioid receptors throughout the central nervous system.

Partial agonists (such as buspirone, aripiprazole, buprenorphine, or norclozapine) also bind and
activate a given receptor, but have only partial efficacy at the receptor relative to a full agonist,
even at maximal receptor occupancy. Agents like buprenorphine are used to treat opiate
dependence for this reason, as they produce milder effects on the opioid receptor with lower
dependence and abuse potential.
An inverse agonist is an agent that binds to the same receptor binding-site as an agonist for that
receptor and inhibits the constitutive activity of the receptor. Inverse agonists exert the opposite
pharmacological effect of a receptor agonist, not merely an absence of the agonist effect as seen
with antagonist. An example is the cannabinoid inverse agonist rimonabant.

A co-agonist works with other co-agonists to produce the desired effect together. NMDA receptor
activation requires the binding of both glutamate, glycine and D-serine co-agonists.
An irreversible agonist is a type of agonist that binds permanently to a receptor through the
formation of covalent bonds. A few of these have been described
A selective agonist is selective for
a specific type of receptor. E.g.
buspirone is a selective agonist for
serotonin 5-HT1A.

The potency of an agonist is

inversely related to its EC50
(Effective Concentration)value.
The EC50 can be measured for a
given agonist by determining the
concentration of agonist needed to
elicit half of the maximum biological response of the agonist. The EC50 value is useful for
comparing the potency of drugs with similar efficacies producing physiologically similar effects.
The smaller the EC50 value, the greater the potency of the agonist, the lower the concentration of
drug that is required to elicit the maximum biological response.

ANTAGONIST:

A receptor antagonist is a type of receptor ligand or drug that blocks or dampens agonist-mediated
responses rather than provoking a biological response itself upon binding to a receptor. In
pharmacology, antagonists have affinity but no efficacy for their cognate receptors, and binding
will disrupt the interaction and inhibit the function of an agonist or inverse agonist at receptors.
Antagonists mediate their effects by binding to the active (orthosteric = right place) site or to
allosteric (= other place) sites on receptors, or they may interact at unique binding sites not
normally involved in the biological regulation of the receptor's activity. Antagonist activity may
be reversible or irreversible depending on the longevity of the antagonist–receptor complex,
which, in turn, depends on the nature of antagonist–receptor binding. The majority of drug
antagonists achieve their potency by competing with endogenous ligands or substrates at
structurally defined binding sites on receptors.

The biochemical definition of a receptor antagonist was introduced by Ariens and Stephensonin
the 1950s. The current accepted definition of receptor antagonist is based on the receptor
occupancy model. It narrows the definition of antagonism to consider only those compounds with
opposing activities at a single receptor. Agonists were thought to turn "on" a single cellular
response by binding to the receptor, thus initiating a biochemical mechanism for change within a
cell. Antagonists were thought to turn "off" that response by 'blocking' the receptor from the
agonist. This definition also remains in use for physiological antagonists, substances that have
opposing physiological actions, but act at different receptors. For example, histamine lowers
arterial pressure through vasodilation at the histamine H1 receptor, while adrenaline raises arterial
pressure through vasoconstriction mediated by alpha-adrenergic receptor activation.

The potency of an antagonist is usually defined by its EC50 value. This can be calculated for a
given antagonist by determining the concentration of antagonist needed to elicit half inhibition of
the maximum biological response of an agonist. Elucidating an EC50 value is useful for comparing
the potency of drugs with similar efficacies, however the dose-response curves produced by both
drug antagonists must be similar. The lower the EC 50 the greater the potency of the antagonist, and
the lower the concentration of drug that is required to inhibit the maximum biological response.
Lower concentrations of drugs may be associated with fewer side-effects.
Affinity
The affinity of an antagonist for its binding site (Ki), i.e. its ability to bind to a receptor, will
determine the duration of inhibition of agonist activity.

Types
Competitive
Competitive antagonists (also known as surmountable antagonists) reversibly bind to receptors at
the same binding site (active site) as the endogenous ligand or agonist, but without activating the
receptor. Agonists and antagonists "compete" for the same binding site on the receptor. Once
bound, an antagonist will block agonist binding. The level of activity of the receptor will be
determined by the relative affinity of each molecule for the site and their relative concentrations.
High concentrations of a competitive agonist will increase the proportion of receptors that the
agonist occupies, higher concentrations of the antagonist will be required to obtain the same degree
of binding site occupancy. In functional assays using competitive antagonists, a parallel rightward
shifts of agonist dose–response curves with no alteration of the maximal response is observed.
The interleukin-1 receptor antagonist, IL-1Ra is an example of a competitive antagonist. The
effects of a competitive antagonist may be overcome by increasing the concentration of agonist.
Often (though not always) these antagonists possess a very similar chemical structure to that of
the agonist.
Non-competitive
The term "non-competitive antagonism" (sometimes called non-surmountable antagonists)can be
used to describe two distinct phenomena: one in which the antagonist binds to the active site of
the receptor, and one in which the antagonist binds to an allosteric site of the receptor. While the
mechanism of antagonism is different in both of these phenomena, they are both called "non-
competitive" because the end-results of each are functionally very similar. Unlike competitive
antagonists, which affect the amount of agonist necessary to achieve a maximal response but do
not affect the magnitude of that maximal response, non-competitive antagonists reduce the
magnitude of the maximum response that can be attained by any amount of agonist. This property
earns them the name "non-competitive" because their effects cannot be negated, no matter how
much agonist is present. In functional assays of non-competitive antagonists, depression of the
maximal response of agonist dose-response curves, and in some cases, rightward shifts, is
produced. The rightward shift will occur as a result of a receptor reserve (also known as spare
receptors) and inhibition of the agonist response will only occur when this reserve is depleted.
An antagonist that binds to the active site of a receptor is said to be "non-competitive" if the bond
between the active site and the antagonist is irreversible or nearly so. This usage of the term "non-
competitive" may not be ideal, however, since the term "irreversible competitive antagonism" may
also be used to describe the same phenomenon without the potential for confusion with the second
meaning of "non-competitive antagonism" discussed below.
The second form of "non-competitive antagonists" act at an allosteric site. These antagonists bind
to a distinctly separate binding site from the agonist, exerting their action to that receptor via the
other binding site. They do not compete with agonists for binding at the active site. The bound
antagonists may prevent conformational changes in the receptor required for receptor activation
after the agonist binds. Cyclothiazide has been shown to act as a reversible non-competitive
antagonist of mGluR1 receptor.

Uncompetitive
Uncompetitive antagonists differ from non-competitive antagonists in that they require receptor
activation by an agonist before they can bind to a separate allosteric binding site. This type of
antagonism produces a kinetic profile in which "the same amount of antagonist blocks higher
concentrations of agonist better than lower concentrations of agonist".Memantine, used in the
treatment of Alzheimer's disease, is an uncompetitive antagonist of the NMDA receptor.

Silent antagonists
Silent antagonists are competitive receptor antagonists that have zero intrinsic activity for
activating a receptor. They are true antagonists, so to speak. The term was created to distinguish
fully inactive antagonists from weak partial agonists or inverse agonists.