ION CHANNELS

Ion channels are pore-forming membrane proteins whose function is

- 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 membranes,
- regulating cell volume.
Their activation translates into a rapid physiological effect.
 ION CHANNELS: general concepts

Channels differ with respect to the ion they let pass (Na+, K+, Ca++, Cl−).

The ion channel selectivity discriminates cations (Na, K, Ca) from anions (Cl),
and allow selectivity among cations (Na, K, Ca).
 ION CHANNELS: mechanisms of selectivity

Despite the small differences in their

dimension, ions rarely go through
the "wrong" channel. For example,
sodium or calcium ions rarely pass
through a potassium channel.

K+ in H2O Na+ in H2O

One hypothesis about selectivity
considers that hydrated ions behave
differentially and postulates that the pore
lining could efficiently replace the water
molecules that normally shield specific
ions. Based on this, potassium ion are K+ inside pore Na+ inside pore
allowed in certain channels; conversely,
sodium ions are too small to allow such
shielding, and therefore could not pass
through.
 ION CHANNELS: physiological effects

Intracellular compartment
GATING THE FLOW OF IONS ACROSS
THE CELL MEMBRANE MODIFIES
THE MEMBRANE POTENTIAL AND
THE CELL PHYSIOLOGICAL STATE

Membrane potential is the difference in

electric potential between the interior
and the exterior of a biological cell.

With respect to the exterior of the cell,

typical values of membrane potential
range from –40 mV to –80 mV.

Extracellular compartment
 ION CHANNELS: structure

subunits
4 or subunits Each polipeptidic subunit The M2 region of
5 closely has 4 hydrophobic regions each subunit
around packed (M1-M4) spanning the cell each others
faces on the
filled porea through
water- membrane inner side of the
the plane of the channel and
membrane or lipid determines ion
bilayer selectivity
 ION CHANNELS: ROC and VOC

• Ion entry cells

into neurons)
(particularly
occurs either
mainly through
operated channels receptor-
(ROC)
or voltage-
operated channels
(VOC).

•The function of ROC depends crucially on the action of agonists, antagonists or

compounds modulating particular types of receptors (GABA A, NMDA, ACh N
receptors).

•The function of VOC is closely connected with the activity of protein kinases
and the processes of phosphorylation of membrane proteins (K+, Na+, Ca2+
channels).
 Receptor-operated (ROC) CHANNELS

•PERMEABILITY to CATIONS LIGAND RECEPTOR

(Na+, Ca++, K+)
Acetilcholine Nicotinic R

Ion entry produces Glutamate and other excitatory NMDA R

aa AMPA
DEPOLARIZATION R KAR
(cell activation)
Serotonin 5-HT3 R
ATP and purines P2X

Ciclic nucleotides CNG

(cAMP and cGMP)

• PERMEABILITY to ANIONS LIGAND RECEPTOR

(Cl-)
Ion entry produces GABA GABAA
HYPERPOLARIZATION Glicine Gly R
(cell inhibition)
 Acetylcholine
Acetylcholine (ACh) is the neurotransmitter of cholinergic system.
These nerve cells are activated by or contain and release acetylcholine

The brain cholinergic system has

been associated with a number of
cognitive functions, including
ACh is the preganglionic neurotransmitter memory, selective attention, and
for both the sympathetic emotional processing.
parasympathetic nervous system, and the
and
postganglionic neurotransmitter in the
parasympathetic nervous system
 ACH RECEPTORS
Acetylcholine binds to both muscarinic and nicotinic receptors.

NICOTINIC R MUSCARINIC R
NN, NM M1, M2, M3, M4, M5
Nicotinic receptors are Receptor- Muscarinic receptors are G-protein
operated channel receptors and get their coupled receptors and get their name
name from nicotine, which selectively from a chemical that selectively attaches
binds to the nicotinic receptor.[ to that receptor, called muscarine.
 ROC: Nicotinic receptor

• 5 transmembrane subunits:
α (2), β, γ, δ or ε
• Each subunit possesses 4 TSM
•They form a pentameric
structure, with a γ subunit
interposing the 2 α subunits

•MUSCULAR nicotinic receptor is a

cation channel allowing cell entrance
of NA, and to a lesser extent , of K
and Ca
Muscular Neuronal • NEURONAL nicotinic
receptor
preferentially permits Ca entry
ROC: Nicotinic receptor

•The 2 α subunits represent the BINDING

SITE for the ligand (Ach). Both subunits
must be occupied by Ach to allow receptor
activation. The first binding facilitates the
second (cooperation)

•GATING: Binding of the 2 Ach molecules

induces a conformational change that
opens the channel.
 ROC: Nicotinic receptor

•The M2 sequence of each

TSM is a segment enriched in
Ser or Thr (negatively charged)
aminoacids, forming three
rings.
1
• These three consecutive rings
represent the SELECTIVITY
2 FILTER facilitating the entry of
cations and excluding anions.
3

• The cytoplasmic region of the receptor includes a REGULATORY P SITE

that can be modulated by phosphorylation.
• At this level additional regulatory sites have the role to anchor the
receptor to specific regions of the cell membrane
 Glutamate
L-Glutamate is the major excitatory neurotransmitter in the mammalian CNS.
L-glutamate is responsible for basal excitatory synaptic transmission and many forms of
synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD),
mechanisms that are thought to underlie learning and memory.

Glutamate is contributing to normal neural transmission, development,

differentiation, and plasticity.
 Glutamate RECEPTORS
L-glutamate acts via two classes of receptors, ligand gated ion channels ( ionotropic
receptors, iGluR) and G-protein coupled (metabotropic, mGluR) receptors.

iGlut Receptors mGlut

Receptors

iGluR are named after their respective

more specific agonist: mGlutR are G-protein coupled
receptors and trigger a second
- NMDA R, N-methyl-D-aspartate messenger cascade.
- AMPA R, α-amino-3-hydroxy-5-methyl-4- They are found both at the pre-
isoxazole propionic acid and post-synaptic neurons
- KA R, kainate
 ROC: AMPA-R
•AMPA receptors mediate fast synaptic transmission in the CNS and are
composed of subunits GluA1-4. Like all the iGlu R subunits, GluA subunits have an
extracellular N-terminus and an intracellular C-terminus. The ligand binding
domain is made up from N-terminal region S1 and S2.

GluA2 subunit
•The C-terminus of
GluA2 subunit
binding sites the
for
number contains
a
proteins. The effects oflarge
these
protein-protein
of interactions
is crucial in localisation and
trafficking ofinteracting
these receptors
so that they can fulfill their
roles in plasticity.

AMPA R are permeable mostly to Na and, to a lesser extent, to Ca ions.

•The calcium permeability of the GluA2 subunit is determined by the so called Q/R
post-transcriptional editing site - GluA2(Q - glutamine) is calcium permeable whilst
GluA2(R - arginine) is not.
 ROC: KA R
•AMPA and kainate R are grouped together in the 'non-NMDA receptor' family.
Kainate R are similar to AMPA R, and are formed by multimeric assemblies of
GluK1-3 and GluK4,5 subunits. GluK1-3 subunits can combine to form functional
heteromeric and homomeric assemblies.

•Until relatively recently, KAR

functional and physiological role in
the mammalian CNS was unclear.
They seem present at both sides of
the synapse.
•Pre- and postsynaptic KAR can
regulate transmission at many
synapses in a specific manner, and
seem to be involved in short- and
long-term plastic phenomena.

KA R are permeable to Na and Ca .

However, their kinetics is much lower than AMPA R
 ROC: NMDA R
Unusually for the iGluR, L-GLUTAMATE IS NOT THE ONLY AGONIST FOR THE
NMDA R. Another amino-acid, GLYCINE, IS A CO-AGONIST and both transmitters
must bind to their respective binding site in order for the receptor to function.

The binding sites for glutamate and

glycine are found on different
subunits:
- GLYCINE binds to the GluN1
subunit while
- GLUTAMATE binds to the GluN2
subunit.

The GluN2B subunit also possesses

a BINDING SITE for polyamines,
regulatory molecules that modulate
the functioning of the NMDA
receptor.

NMDA R are highly permeable to Ca and , to a lesser extent, to Na .

 ROC: NMDA R
The NMDA R channel is normally blocked by Mg2+ in a voltage- and use-
dependent manner.

Although necessary, binding of both L-

GLUTAMATE and GLYCINE is still not Mg2+
sufficient to completely activate NMDA
receptor and favor Ca2+ entry

Depolarization of the postsynaptic

neuron via AMPA R activation releases
the Mg2+ block on the NMDA R.

Ca2+
entry is therefore subsequent to
complete post-synaptic depolarization
(required to remove Mg2+). This effect is
obtained by early activation of AMPA R
Glutamate RECEPTORS summary
 Gamma Amino Butyric Acid (GABA)
GABA is the chief inhibitory neurotransmitter in the mammalian central
nervous system. It plays the principal role in reducing neuronal excitability
throughout the nervous system. In humans, GABA is also directly responsible
for the regulation of muscle tone.
 GABA RECEPTORS
GABA binds to both GABAA and GABAB receptors.

GABA A Receptor GABA B Receptor

GABAA receptors are ligand-gated GABAB receptors are members of the 7-

channel receptors TM G protein-coupled receptors
 ROC: GABAA RECEPTOR
•The γ-aminobutyric acid, type A (GABAA) receptor is a chloride-conducting
receptor composed of α, β, and γ subunits assembled in a pentameric structure
forming a central pore.
•The majority of GABAA Rs are believed to be expressed as heteromeric complexes of
2α, 2β, and 1 γ subunit
•The 2β subunits
represent the BINDING
SITE for the ligand
(GABA).

• Both subunits must be

occupied by GABA
allow to receptor
activation. The
binding facilitates
the second first
(cooperation)
 ROC: GABAA RECEPTOR
• Each subunit has a large extracellular agonist • The M2 sequence of
binding domain and four each TSM forms the
transmembrane domains (M1–M4), SELECTIVITY FILTER
with
transmembrane (M2)
the
domain second
lining the pore. facilitating the entry of
Cl-
anions.

• The αsubunits represent

the SITE, a
ALLOSTERIC
modulatory where
binding
region of ligands different
from may
facilitate/obstacolate
GABA
GABA/RECEPTOR interaction. the

Cl-
• Another ligand site is present on the deep part of the channel, on the β subunit.
Binding on this site allows a different modulation of channel opening that may
excede the GABA-mediated effects
 Voltage-operated (VOC) CHANNELS
The function of VOC is closely dependent on transmembrane electrochemical
gradient. A gradient is represented by the different concentration of ions on
either side of the membrane. The open conformation of the ion channel allows
for the translocation of ions across the cell membrane, while the closed
conformation does not. The activity of these channels is connected with the
activity of enzymes and the processes of phosphorylation of membrane proteins.

Post-synaptic membrane Muscle membrane

with ROC’s with VOC’s
 VOC CHANNELS
Electrophysiological studies indicate the existence of three main types of VOC
(K+, Na+, Ca2+ channels). In number of neurons various subtypes of Ca2+ channels
(P, T, N and L-type) occur together.

Na+ channels

Ca++ channels

K+ channels
 VOC CHANNELS
In each protein subunit, the membrane-spanning segments, designated S1-
S6, all take the form of alpha helices with specialized functions.
The S1-S4 TMS serve as the voltage-sensing region.
The S5-S6 TMS and pore loop have a key role in ion conduction, and
represent the gate and pore of the channel.

S1-4
Voltage – S5-6
sensing region Pore region

S1 S6

S4 C- terminal
I II III IV
N- terminal
 VOC CHANNELS: state of activity
A voltage gated ion channel can be in three states: closed, open or inactivated.

The inactive state, which is stable and non-conducting, is caused by the physical
blockage of the pore. The blockage is caused by a “ball” of amino acids attached
to the main protein by a string of residues on the cytoplasmic side. The ball
enters the open channel and binds to the hydrophobic inner vestibule at the
center of the channel.
The blockage causes inactivation of the channel by stopping the flow of ions.
 Na+ CHANNELS
VOC Na+ channels are formed by a single subunit and are expressed in
all excitable tissues.

Na+ channels are responsible for the

rapid membrane depolarization during the action potential.
Activation and inactivation are voltage-dependent, very fast processes (1-10
ms). During the inactivation state, the channel is in a refractory period.
 VOC Na+ CHANNELS

Na+ channels are important drug targets. Drugs bind the Na channel inside the pore.
Their binding maintains the channel in the inactivated state. Since they reach their
binding site when the channel is open, their inhibitory effect is use- and voltage-
dependent.
Drugs acting as Na+ channels blockers include Class I anti-arrhythmics,
anticonvulsants, local anesthetics
 Non-VOC Na+ CHANNELS
The epithelial Na+ channels (ENaC) are non-voltage gated, highly Na-selective
channels. ENaC activity is rate limiting for Na+ reabsorption in the distal nephron. The
long term control of blood pressure involves Na+ homeostasis through the precise
regulation of ENaC in the aldosterone-sensitive distal nephron

Inhibition of these channels results in reduced K excretion. This effect is obtained by

some potassium-sparing diuretics such as amiloride
 K+ CHANNELS
K+ channels are found in virtually all living organisms. They form K+ -selective pores
that span cell membranes and conduct rapidly and selectively K+ ions down their
electrochemical gradient.
These channels act to set or reset the resting potential in many cells.

Voltage-gated K+ channels
Calcium-activated K+ channels

Inward rectifier K+ channels “Leak” or background K+ channels

K+ CHANNELS: structure

2TM/P channels (which consist of two transmembrane

(TM) helices with a P loop between them), exemplified
by inwardly rectifying K+ channels.
Channel is formed by (2TM/P x4) tetramer

6TM/P channels, which are the predominant class

among ligand-gated and voltage-gated K+ channels.
Channel is formed by (6TM/P x4) tetramer

4TM/2P channels, which consist of two repeats of

2TM/P channels. 4TM/2P channels are far more
common than was originally thought.
Channel is formed by (2TM/P x2) dimer
 K+ CHANNELS

Voltage-gated K+ channels Kv channels are one of the key components in

generation and propagation of electrical impulses
in nervous system and in the heart. Upon changes
in transmembrane potential, these channels open
and allow passive flow of K+ ions from the cell to
restore the membrane potential. They are the
target of Class III anti-arrhythmic drugs

KCa channels can be grouped into three distinct

Calcium-activated K+ channels
subfamilies,
• large-conductance (BK),
• intermediate conductance (IK),
• small conductance (SK) channels.
These channels are activated largely in response
to calcium influx during action potentials.
Activation of these channels is to hyperpolarize
the membrane.
 K+ CHANNELS
Inward rectifier K+ channels
include several subfamilies, among which the most
KIR
important are
• IRK (strong inward rectifier K+ channels)
• GIRK (G-protein-activated rectifier K+
inward
channels)
• (ATP-sensitive K+ channels)
KATP
These channels are responsible for repolarizing a cell
following an action potential
Kir close upon depolarization, slowing membrane
CARDIAC MYOCYTES repolarization and helping maintain a more prolonged
cardiac action potential.
ENDOTHELIAL CELLS Kir are involved in regulation of nitric oxide synthase.
KIDNEYS Kir export surplus K+ into collecting tubules for removal in
the urine, or reuptake K+ back into the body.
G-protein activated IRKs (Kir3) are important regulators,
NEURONS AND HEART modulated by neurotransmitters.

PANCREAS BETA CELLS KATP channels control insulin release.

 K+ CHANNELS
“Leak” or background K+ channels

This type of potassium channel is formed by

two homodimers that create a channel that
leaks potassium out of the cell
They are all voltage-independent and can be
opened by heat, membrane stretching,
intracellular acidosis, and certain anesthetics.
These channels are responsible for a high K+
conductance under basal state conditions and
therefore contribute to resting potential

•TRAAK channels are mechanically activated when there is a convex curvature

in the membrane that alters the channel’s activity (membrane stretching)
•TASK channels are sensitive to changes in extracellular pH and inhibited by
extracellular acidification
•TALK channels are primarily expressed in the pancreas, and activated at
alkaline pH
 Calcium
Calcium is an ubiquitous intracellular messenger, controlling cellular processes ranging
from gene transcription, muscle contraction and cell proliferation

The Ca2+ signaling apparatus involves various channels, pumps, and transporters
 Calcium regulatory mechanisms
CALCIUM ENTRY (“ON”) MECHANISMS
VDCCs -voltage-dependent calcium channel; ROC-Ligand-gated channels, SOCE -store-operated
calcium entry; TRP -transient receptor potential channels; ASIC -acid-sensing ion channels; IEIC -
inward excitotoxic injury current calcium-permeable channels; NCX -sodium-calcium exchanger
(operating in entry mode).
CALCIUM INTRACELLULAR SEQUESTERING AND RECYCLING MECHANISMS
SERCA -Sarcoplasmic-Endoplasmic Reticulum Ca2+-ATPase; Ryr –ryanodine receptors.
CALCIUM EXIT (“OFF”) MECHANISMS
PMCA -Calcium ATPase pump; NCX sodium-calcium exchanger
(operating in exit mode).
 CALCIUM “ON” MECHANISMS: VOCCs
Voltage-Operated Calcium Channels are slightly permeable to Na (also called Ca2+-
Na+ channels), but their permeability to Ca2+ is about 1000-fold greater. At resting
membrane potential, VOCCs are normally closed. They are activated (i.e., opened)
at depolarized membrane potentials and this is the source of the "voltage-
dependent" definition.

Activation of particular
VOCCs allows Ca2+ to rush
into the cell, which,
depending on the cell type,
results in activation of
calcium-sensitive potassium
channels,
muscular contraction,
neurons, up-excitation of
regulation
gene expression, or release
of
of hormones
or neurotransmitters.
 Voltage-operated Ca++ CHANNELS (VOCCs)
TYPE VOLTAGE MOST OFTEN FOUND IN
Skeletal muscle, smooth muscle, bone
(osteoblasts), ventricular myocytes (responsible
L-TYPE CALCIUM CHANNEL HVA for prolonged action potential in cardiac cell;
("LONG-LASTING" AKA "DHP (high voltage also termed DHP receptors), dendrites and
RECEPTOR") activated) dendritic spines of cortical neurones
L-Type channel blockers are used as
antihypertensive and antiarrhythmic drugs

P-TYPE CALCIUM CHANNEL HVA

("PURKINJE") /Q-TYPE (high voltage Purkinje neurons in the cerebellum / Cerebellar
CALCIUM CHANNEL activated) granule cells

N-TYPE CALCIUM CHANNEL HVA Throughout the brain and peripheral nervous
("NEURAL"/"NON-L") (high-voltage- system.
activated)

R-TYPE CALCIUM CHANNEL intermediate-

("RESIDUAL") voltage- Cerebellar granule cells, other neurons
activated
neurons, cells that have pacemaker activity,
bone (osteocytes)
T-TYPE CALCIUM CHANNEL low-voltage- T-Type channel blockers are used as
("TRANSIENT") activated antiepileptic and neuropathic painkiller drugs
 CALCIUM INTRACELLULAR RECYCLING MECHANISMS

Store-operated Ca++ Channels (SOCC) Transient-receptor-potential Channels (TRP)

SOCC can be activated by any procedure activated by cAMP, cGMP, Arachidonic acid,
that empties the stores sphingosine, ADPribose
Receptor-operated Ca++ Channels (ROC)
• nACh R InsP3-Receptor-Ca2+-Channels
• NMDA R, kainate R allow Ca2+ release form intracellular stores
• 5HT3
• P2X

Ryanodine receptors (RYR)

activated by cADPR
• Ryr1 (muscle cells)
• Ryr2 (heart)
• Ryr3 (neurons)
 CALCIUM “OFF” MECHANISMS

Na+/Ca2+ exchanger (NCX1-3) PMCA (Plasma Membrane Ca2+ ATPase)

is an antiporter the plasma membrane pump is powered by the
membrane
protein that removes Ca2+ using hydrolysis of ATP, with a stoichiometry of 1 Ca2+
the from ion/1 molecule of ATP hydrolysed. High affinity
energy
electrochemical the but low speed. Its activity is modulated by the
gradient
Na+. The NCX removes of
1 single calmodulin (CaM) protein (Ca/ATP 1:1)
Ca2+
ion in exchange for the
import of 3 Na+ ions. Low
affinity but high speed (Ca/Na
1:3)

SERCA (Sarcoplasmic-
Endoplasmic Reticulum Ca2+-
ATPase)
It is a Ca2+ ATPase that transfers
Ca2+ from the cytosol of the cell
to the lumen of the SR at the
expense of ATP hydrolysis
during muscle relaxation.
The pump transports 2 Ca2+
ion/1 molecule of ATP