Ion Channels

Voltage-gated Channels
Ligand-gated Channels
TRP Channels
Introduction
 The lipid bilayer of the plasma membrane is impermeable to
anions and cations, yet changes in the flux of ions across the
plasma membrane are critical regulatory events in both
excitable and non-excitable cells.
 To establish and maintain the electrochemical gradients
required to maintain a membrane potential, all cells express ion
transporters for Na+, K +, Ca2+, and Cl− .
 For example, the Na+ ,K+ -ATPase pump expends cellular
ATP to pump Na+ out of the cell and K+ into the cell .
 The electrochemical gradients thus established are used by
excitable tissues such as nerve and muscle to generate and
transmit electrical impulses, by non-excitable cells to
trigger biochemical and secretory events, and by all cells to
support a variety of secondary symport and antiport
processes.
 Passive ion fluxes down cellular electrochemical gradients are
regulated by a large family of ion channels located in the
membrane.
 Humans express ∼232 distinct ion channels to precisely
regulate the flow of Na+, K+, Ca2+, and Cl− across the cell
membrane.
 Because of their roles as regulators of cell function, these
proteins are important drug targets.
 The diverse ion channel family can be divided into sub
families based on the mechanisms that open the channels, their
architecture, and the ions they conduct.
 They can also be classified as voltage-activated, ligand-
activated, store-activated, stretch-activated, and temperature-
activated channels.
 Examples of channels that are major drug targets are detailed
next.
Voltage-gated ion channel
superfamily
Voltage-Gated Channels
 Humans express multiple isoforms of voltage-gated channels
for Na+, K+, Ca2+, and Cl− ions.
 Voltage-activated Na+ channels
 In nerve and muscle cells, voltage-gated Na+ channels are
responsible for the generation of robust action potentials that
depolarize the membrane from its resting potential of −70 mV
up to a potential of +20 mV within a few milli seconds.
 These Na+ channels are composed of three subunits, a pore-
forming α subunit and two regulatory β subunits. The α subunit
is a 260 kDa protein containing four domains that form a Na+
ion-selective pore by arranging into a pseudo-tetramer shape.
The β subunits are ∼36 kDa proteins.
  Each domain of the α subunit contains six membrane-spanning
helices (S1-S6) with an extracellular loop between S5 and S6,
termed the pore-forming or P loop; the P loop dips back into the
pore and, combined with residues from the corresponding P loops
from the other domains, provides a selectivity filter for the Na+
ion.
 Four other helices surrounding the pore (one S4 helix from each
of the domains) contain a set of charged amino acids that form the
voltage sensor and cause a conformational change in the pore at
more positive voltages leading to opening of the pore and
depolarization of the membrane.
 Segments (S5 and S6) and the pore loop were found to be responsible for ion conduction.

Transmembrane Lipid bilayer

segment (cylinder)

Voltage sensor
part of the channel Pore
General architecture of voltage-gated channels (Na+ and Ca2+).The “+” or “-“ signs
indicate charges that have been implicated in voltage sensing.
 The voltage-activated Na+ channels in pain neurons are
targets for local anesthetics such as lidocaine and tetracaine,
which block the pore, inhibit depolarization, and thus
block the sensation of pain.
 They are also the targets of the naturally occurring marine
toxins, tetrodotoxin and saxitoxin.
 Voltage-activated Na+ channels are also important targets of
many drugs used to treat cardiac arrhythmias.
A. Diagram of a voltage-activated Na+ channel with the pore in the open and
closed state.
The P loops are shown in blue, angled into the pore to form the selectivity filter.
The S4 helices forming the voltage sensor are shown in orange, with the
positively charged amino acids displayed as red dots.
B. Ligand-gated nicotinic acetylcholine receptor expressed in the skeletal
muscle neuromuscular junction. The pore is made up of five subunits, each
with a large extracellular domain and four transmembrane helices (one of
these subunits is shown at the left of panel B.
The helix that lines the pore is shown in blue.
The receptor is composed of 2 α subunits, and β, γ, and δ subunits.
Voltage-gated Ca2+channels
 Voltage-gated Ca2+ channels have a similar architecture to
voltaget-gated Na+ channels with a large α subunit (four
domains of six membrane-spanning helices) and three
regulatory subunits (the β, δ and γ subunits).
 There are multiple isoforms of these channels that are widely
expressed in nerve, cardiac and smooth muscle cells.
 Ca2+ channels can be responsible for initiating an action
potential (as in the pacemaker cells of the heart), but are more
commonly responsible for modifying the shape and duration of
an action potential initiated by fast voltage-gated Na+ channels.
 These channels initiate the influx of Ca2+ that stimulates the
release of neurotransmitters in the central, enteric, and
autonomic nervous systems, and that control heart rate and
impulse conduction in cardiac tissue.
 The L-type voltage-gated Ca2+ channels are subject to
additional regulation via phosphorylation by PKA.
 Thus, when the sympathetic nervous system releases
norepinephrine onto β adrenergic receptors in cardiac tissue,
raising cAMP and activating PKA, the phosphorylated L-type
channels allow more Ca2+ to flow into the cytoplasm,
increasing the force of contraction.
 Voltage-gated Ca2+ channels expressed in smooth muscle
regulate vascular tone; the intracellular concentration of Ca2+
is critical to regulating the phosphorylation state of the
contractile apparatus via the activity of the Ca2+/calmodulin-
sensitive myosin light chain kinase.
 Accordingly, the Ca2+ channel antagonists such as nifedipine,
diltiazem, and verapamil are effective vasodilators and are
widely used to treat angina, cardiac arrhythmias, and
hypertension.
Voltage-gated K+ channels
 Voltage-gated K+ channels are the most numerous and
structurally diverse members of the voltage-gated channel
family.
 Humans express ∼78 distinct K+ channels and nearly all of
them are voltage-gated.
 The voltage-gated Kv channels form channels as tetramers
with topology similar to the Na+ and Ca2+ channels, but
rather than having four domains, they consist of four separate
subunits that each incorporate six membrane-spanning
domains.
 All these channels are expressed in nerve, cardiac tissue,
skeletal and smooth muscle, as well as non-excitable tissues.
 Increasing K+ conductance through these channels drives the
membrane potential more negative; thus, these channels are
important in regulating resting membrane potential and
resetting the resting membrane at −70 to −90 mV following
depolarization.
 Some forms of epilepsy are caused by natural mutations in Kv
channels, and drugs such as retigabine that favor opening of
Kv channels are under study for the treatment of epilepsy.
 The cardiac KCNH2 channel , known as hERG (human ether-
a-go-go-related gene), is responsible for hereditary as well as
acquired (drug -induced) long QT syndrome.
 It is also the primary target of many anti-arrhythmic drugs
that prolong repolarization.
 Ligand-gated Channels
 Channels activated by the binding of a ligand to
a specific site in the channel protein have a
diverse architecture and set of ligands.
 Major ligand gated channels in the nervous
system are those that respond to excitatory
neurotransmitters such as acetylcholine or
glutamate (or agonists such as AMPA and
NMDA) and inhibitory neurotransmitters such
as glycine or γ-aminobutyric acid (GABA).
 Activation of these channels is responsible for
the majority of synaptic transmission by
neurons both in the CNS and in the periphery.
 AMPA: α-amino-3-hydroxy-5-methyl-4-
isoxazole-propionate
 In addition, there are a variety of more specialized ion
channels that are activated by intracellular small molecules,
and are structurally distinct from conventional ligand-gated
ion channels.
 These include ion channels that are formally members of the
Kv family, such as the hyperpolarization and cAMP-gated
(HCN) channel expressed in the heart that is responsible for
the slow depolarization seen in phase 4 of AV and SA nodal
cell action potentials and the cyclic nucleotide-gated (CNG)
channels important for vision.
 Hyperpolarization-activated cyclic nucleotide–gated
(HCN) channels
 The intracellular small molecule category of ion channels also
includes the IP-sensitive Ca2+ channel responsible for release of
Ca2+ from the ER and the sulfonylurea receptor” (SUR1) that
associates with the Kv channel to regulate the ATP dependent K+
channel ( ATP ) In pancreatic beta cells.
 The K ATP channel is the target of oral hypoglycemic drugs such
as sulfonylureas and meglitinides that stimulate insulin release
from pancreatic β-cells and are used to treat type 2 diabetes.
 Other specialized channels include the 5-HT3-regulated channel
expressed on afferent vagal nerves that stimulates emesis.
 Ondansetron is an important antagonist of the 5-HT3-gated
channel used to inhibit emesis caused by drugs or disease.
 The nicotinic acetylcholine receptor provides an instructive
example of a ligand-gated ion channel.
 Isoforms of this channel are expressed in the CNS, autonomic
ganglia and at the neuromuscular junction.
 The pentameric channel consists of four different subunits (2α,
β, δ, γ) in the neuromuscular junction or two different subunits
(2α,3β) in autonomic ganglia.
 Each α subunit has an identical acetylcholine binding site; the
different compositions of the other three subunits between the
neuronal and neuromuscular junction receptors accounts for
the ability of competitive antagonists such as rocuronium to
inhibit the receptor in the neuromuscular junction without
effect on the ganglionic receptor.
 This property is exploited to provide muscle relaxation during
surgery with minimal autonomic side effects.
 Each subunit of the receptor contains a large, extracellular
N-terminal domain, four membrane-spanning helices (one of
which lines the pore in the assembled complex), and an
internal loop between helices 3 and 4 that forms the
intracellular domain of the channel .
 The pore opening in the channel measures ∼3 nm whereas the
diameter of a Na+ or K+ ion is only 0.3 nm or less.
 Accordingly, ligand-gated ion channels do not possess the
exquisite ion selectivity found in most voltage-activated
channels and activation of the nicotinic acetylcholine receptor
allows passage of both Na+ and K+ ions.
 The major excitatory transmitter at CNS synapses is glutamate.
 There are three types of ionotropic glutamate receptors (AMPA,
NMDA, and kainate), named after the ligands that selectively
activate them.
 They have a topology similar to that of the nicotinic acetylcholine
receptor: the channel is made up of five subunits organized with a
large extracellular region, a pore, and a small intracellular face.
 Activation of these channels with glutamate markedly increases
Na+ and K+ conductance leading to depolarization.
 NMDA receptors are less ion-selective; activation increases Na+,
K+, and Ca2+ conductance, with the Ca2+ signal being used for
additional signaling events.
 Over one-third of synapses in the brain are inhibitory; the major
inhibitory transmitters are glycine and γ aminobutyric acid (GABA).
 Glycine and ionotropic GABA A receptors have a topology like
that of the glutamate and nicotinic acetylcholine receptors, with five
subunits (α, β, γ, δ and ρ), a ligand binding domain, and pore-
forming helices.
 Activation of these channels increases Cl- conductance, which
hyperpolarizes the cell membrane and inhibits excitability.
 GABA A receptors are targets of important sedative-hypnotic drugs
such as the benzodiazepines and barbiturates, and are also important
in the mechanisms of ethanol and general anesthetics.
TRP Channels.
 The transient receptor potential (TRP) channels comprise a
superfamily of ion channels that is remarkable in its diversity
and domain structure.
 Although the TRP channels are not presently targets of
approved drugs, there is significant interest in developing
drugs that can alter the function of these ion channels because
of their roles in various sensory phenomena such as pain,
temperature, osmolarity, touch, olfaction, vision, and hearing.
 Because these channels contain multiple domains, they can act
as signal integrators and most can be activated by multiple
mechanisms.
 There are 27 TRP channel genes in humans, representing six
different TRP channel families.
 TRP channels contain six membrane-spanning segments and
the functional ion channels consist of tetrameric complexes.
 Closely related TRP channels can form heterotetramers.
 The TRP channels are cation channels, but as with other
heteromultimeric ion channels, the subunit composition of the
multimeric channels can prescribe a number of important
channel characteristics, including ion selectivity and activation
properties.
 The intracellular domains of TRP channels can include
ankyrin domains, protein kinase domains, and ADP-
ribose pyrophosphatase domains.
 Mutations in TRP channels are known to cause several
diseases including hypo-magnesemia and hypo-
calcemia, and various renal disorders and
neurodegenerative diseases.
Questions?