Ion channels-a class of proteins that is ultimately responsible for generating and orchestrating the
electrical signals passing through the thinking brain, the beating heart, and the contracting muscle.
Ion channels are macro-molecular protein tunnels that span the lipid bilayer of the cell membrane.
They are more efficient than enzymes; small conformational changes change a single channel from
closed to open, allowing up to 10 million ions to flow into or out of the cell each second.
Ion channels are usually classified according to the type of ion they allow to pass—sodium, potassium,
calcium, or chloride—although some are less selective.
The direction in which ions move through a channel is governed by electrical and chemical conc.
gradients. ions flow passively through channels down a chemical gradient.
Electrically charged ions also move in an electrical field, just as ions in solution flow to one of the poles
of a battery connected to the solution.
The point at which the chemical driving force and the electrical driving force are exactly balanced is
called the Nernst potential.
The Nernst potentials of the four major ions across the membrane are :
Sodium,+70mV; potassium,--98mV; calcium,+150mV; and chloride,--30 to
–65mV.
When only one type of ion channel opens, it drives the membrane potential of the entire cell towards the
Nernst potential of that particular ion.
When more than one type of ion channel opens, each type pulls the transmembrane potential of the cell
towards its own Nernst potential.
The overall trans-membrane potential at a given moment is therefore determined by which channels are
open and which are closed.
Patch clamp technique:-
The modern way to see an ion channel in action is to use the patch clamp technique.
(Neher and Sakmann developed the patch clamp technique for which they received the Nobel prize in 1991)
A micro- pipette containing a small electrode is pressed against the cell membrane tightly in way that the
electrode isolates and captures all the ions flowing through the 1 to 3 micro m 2 of membrane that is defined by
the circular border of the micropipettes.
In this fashion the ionic current passing through a single ion channel can be collected and measured.
The current passing through the attached patch, detached patch or whole cell can be measured, providing
information about ion channels within the environment of the cell, in isolation from the rest of the cell or over
the entire cell respectively.
Patch clamp technique
Types of ion channels:-
(1) Voltage gated channels—respond to changes in the membrane potential of the cell.
(2) Ligand gated ion channels— (ionotropic receptors.)
They are membrane receptors which are coupled directly to an ion channel.
The receptor is formed of subunits, and the channel is an integral part of the receptor complex.
These channels are insensitive or are weakly sensitive to membrane potential.
Activation of these channels typically results in a brief (a few milliseconds) opening of the channel.
Ligand binding channels are mainly involved in fast synaptic transmission.
(3) Metabotropic receptors—
Metabotropic receptors act via G-proteins and modulate voltage gated ion channels.
The G-protein is a membrane protein comprising of three sub units(alpha, beta and gamma)
The G-protein can interact directly with the ion channel and this interaction can occur entirely within the
membrane–referred to as membrane delimited pathway----mainly calcium and potassium channels are involved
in this type of signaling.
Metabotropic receptors can also modulate voltage gated ion channels less directly by diffusible second
messengers derived from G-proteins.
The well established second messengers are c-AMP, calcium and phosphoinositides and c-GMP.
Whereas membrane delimited actions occur within the microdomains of the membrane, second
messenger mediated effects can occur over considerable distances.
In contrast to the brief effect of ionotropic receptors, the effects of metabotropic receptor activation can
last tens of seconds to minutes.
Molecular structure of ion channels:- (In general)
Most ion-channel proteins are composed of individual subunits or groups of subunits, with each subunit
containing six hydrophobic transmembrane regions, S1 through S6 .
The sodium and calcium channels comprise a single unit(alpha-1) containing four homologus domains each
containing the six transmembrane-spanning motifs.
Voltage-gated potassium channels (Kv) are composed of four separate subunits, each containing a single six-
transmembrane–spanning motif .
The subunits are assembled to form the central pore in a process that also determines the basic properties of
gating and permeation characteristic of the channel type.
The peptide chain (H5 or P loop) between the membrane-spanning segments S5 and S6 projects into and
lines the water-filled channel pore. Mutations in this region alter the permeation properties of the channel.
S4 contains a cluster of positively charged amino acids (lysines and arginines) and is the major voltage
sensor of the ion channel.
Voltage-dependent "fast inactivation" of the channel is mediated by a tethered amino-terminal–blocking
particle (the "ball and chain") that swings in to occlude the permeation pathway.
Voltage gated calcium channel:
Maintain the plateau phase of action potential in cardiac muscle glands and nerve terminals: second
messenger.
Voltage gated ion channels are composed of a major alpha-1 subunit which encloses the ion channel and
other modulatory subunits like alpha2, beta, gamma and delta.
Types:-
L-type:--
long lasting current. conductance of about 25 pS.
Excitation-contraction coupling in cardiac and SM and also SA and AV node
conductivity.
Blocked by CCBs.
Phenylalkylamines—bind at inner side of transmembrane pore probably at the innermost part of the
channel.
Dihydropyridines—bind by slipping into the cleft between domains III and IV.
T-type:--
Transient current. Conductance around 8 pS.
Have a lower activation threshold. Involved in T current and repetitive spikes in thalamic and other neurons.
Blocked by mebefradil, flunarazine and ethosuximide.
N-type:--
12-20 pS conductance.
Only on neurons in CNS, sympathetic and myentric plexus
Blocked by w -- conotoxin.
The most recently discovered family of ion -channel proteins is that containing the inwardly rectifying
potassium-selective channels (Kir, for K channel, inward rectifier).
These determine the transmembrane potential of most cells at rest, because they are open in the steady state.
Kir channels are known as inward rectifiers because they conduct current much more effectively into the cell
than out of it.
The topography of Kir channels resembles that of Kv channels, but the subunits in Kir channels lack the S1
to S4 segments present in Kv channels.
With only two transmembrane-spanning segments, Kir channels have a deceptively simple domain
surrounding the conserved H5 pore.
However, pore formation by different combinations of subunits, direct gating of G proteins, and interactions
with other proteins adds considerable complexity to the behavior of the Kir channels.
shows a subunit containing six transmembrane-spanning motifs, S1 through S6, that forms the core structure
of sodium, calcium, and potassium channels
The circles containing plus signs in S4, the voltage sensor, are positively charged lysine and arginine
residues.
Key residues lining the channel pore (H5) are found between S5 and S6.
The genes for sodium and calcium channels encode a protein containing four repeats of this basic subunit,
whereas the genes for voltage-activated potassium channels (Kv) encode a protein with only a single subunit.
The genes for Kir channels encode a simple subunit structure containing only an H5 (pore) loop between
two transmembrane-spanning segments.
Voltage-gated potassium channels are composed of four separate subunits, each containing a single six-
transmembrane–spanning motif .
shows four such subunits assembled to form a potassium channel.
LIGAND GATED ION CHANNELS:--
The family of ligand gated ion channels are pentamers of homologus subunits each having a molecular mass
of 40,000 to 60,000 daltons.
The indl. Subunits are about 40% identical in their amino-acid sequences suggesting that arose from a
common primordial gene.
Each subunit has an extracellular and an intracellular domain and the complete receptor encloses an ion
channel which is modified by the binding of the ligand to the receptor.
Nicotinic receptor ----
Location—Post-synaptic regions of vertebrate muscle cells and ganglia of the CNS.
Structure and function---
The nicotinic receptor has become the prototype for the ligand gated ion channels.
Pentamer composed of four distinct subunits—alpha, beta, gamma, delta in the stoichometric ratio of
2 :1:1:1.
Nicotinic receptors in the CNS also exist as pentamers with one, two or rarely more subunits.
There are eight subtypes of alpha (2---- 9) and three subtypes of beta( 2—4).
The five subunits are arranged to circumscribe an internally located channel in a fashion similar to petals on
a lily.
The binding sites are found at the subunits interfaces but of the five interfaces only two in muscle (alpha-
gamma) and (alpha-delta) have evolved to bind ligands.
Attachment of the ligand on both the sites is essential to activate the receptor.
Upon activation its intrinsic channel opens for about 1 millisec.
During this interval 50,000 Na+ ions traverse the channel.
This leads to localized depolarization (MEPP). Their summation to form a post-junctional end plate
potential which triggers the muscle contraction.
Several congenital myasthenic syndromes recently have been found to arise from mutations in the muscle
receptor subunits.
NICOTON AGONI ANTAGONISTS TISSUE RESPONSES
IC ST
RECEPTO
R
skeleletal Phenyl d-Tubocurarine Neuromuscula End-plate depolarization,
muscle(NM ) trimethy r Skeletal muscle contraction.
l- -Bungarotoxin junction
ammoni
um
(PTMA)
Nicotin
e
Neuronal Dimethy Hexamethonium. Autonomic Depolarization and firing of post-
(peripheral) lphenyl ganglia ganglionic neuron;
( NN ) Piperazi Trimethaphan. Depolarization of medullary cell
nium Adrenal and secretion of catecholamines.
(DMPP medulla
)
Epibatid
ine
Nicotine
.
Neuronal Nicotine several Brain and Pre-junctional
(CNS) spinal cord. Control of neurotransmitter
Cystine Release.
epibatidi
ne
Gamma – amino butyric acid (GABA) receptor:-
GABA is the inhibitory transmitter modulating IPSP’s.
GABA receptors are divided into two types GABA –A and GABA-B.
GABA-A receptor is a classical ligand ion channel while GABA-B receptor is coupled to G-proteins that
either inhibit calcium channels or activate potassium channels.
GABA–A receptor chloride channel macro-molecular complex has a pentameric structure assembled from
five subunits which are in turn selected from eight polypeptide classes (alpha, beta, gamma, delta, epsilon, pi,
rho, theta ).
Combination of three major subunits alpha, beta, and gamma are essential for normal physiological and
pharmacologic functions.
Senstivity of the receptor—chloride ion channel complex to various ligands is determined by particular
variants of the major subunits.
Isoforms containing alpha-1, beta-2, and gamma-2 subunits are most sensitive to BZDs and are widely
distributed in the CNS .(BZD—1 receptor)
A change in residue 77 of the gamma-2 subunit prevents BZD binding to the receptor ion channel complex.
Role of other subunits is still incompletely understood.
The GABA-A chloride receptor is one of the most versatile drug responsive machines in the [Link] has got
multiple sites of binding through which various agents act.
Drugs affecting GABA-linked chloride channel:-
GABA Endogenous agonist at GABA-A and promotes chloride influx.
Muscimol Agonist at GABA—A site
Bicuculine Competitive antagonist at GABA—A receptor.
Picrotoxin Blocks chloride channel non-competetively:acts on picrotoxin sensitive site.
Benzodiazepines Agonist at an allosteric BZD site—facilitate GABA action. (increased
frequency of chloride channel )
Barbiturates Agonist at an allosteric site(? Picrotoxin site)-----increased duration of
channel opening. High doses—GABA mimetic action.
Beta-carboline Inverse agonist at BZD site—impede GABA action.
(DMCM)
Flumazenil Competitive antagonist at BZD site.
Glutamate receptors:--
Glutamate is the principal excitatory transmitter in the CNS.
They can be both ionotropic or metabotropic.
Ionotropic receptors have a pentameric structure similar to other ionotropic
receptors.
can be further subdivided into three subtypes based on the action of
Selective agonists:
NMDA------- N-methyl D aspartate
AMPA-------- alpha amino-3- hydroxy-5- methyl isoxazole-4-propionate.
K ----------- Kainate.
NMDA receptors are assembled from two types of subunits NR-1 and NR-2,
each of which can exist in [Link] giving rise to diff. receptor isoforms in
the brain.
The NMDA activated channel is highly permeable to sodium, potassium and
calcium ions.
The subunits comprising AMPA and Kainate receptors are termed GLUR 1-7 and
KA 1-2.
The AMPA and Kainate channels are permeable mainly to sodium and potassium ions and are often
grouped together as non-NMDA receptors.
Antagonists—
At NMDA—Ketamine and phencyclidine.
At AMPA and Kainate—CNQX; GYK 152466.
The metabotropic glutamate receptors act indirectly on ion channels via [Link] GPCR either
stimulates PLC or inhibit Adenylyl cyclase.
The NMDA receptors play a critical role in certain forms of learning and memory.
Glutamate release during neuronal injury can by activating the NMDA receptor cause further cell injury and
[Link] may attenuate neuronal damage.
