PHYL3001

Physiology of Membranes, Muscles and Signalling

Ion Channel Structure and Function

Dr Gavin Pinniger
 Objectives

• Describe the structure and function of voltage-gated ion

channels
• Explain the molecular basis for voltage gating, inactivation
and ion channel selectivity
• Understand the similarities and differences between different
classes of ion channels
• Understand the different types of ionic currents and how they
relate to ion channel structure
• Become familiar with the impact of genetic mutations on ion
channel function and how this impact membrane potentials
and cell function
Voltage-gated–like (VGL) ion channel
superfamily

Dendrogram based on
evolutionary relationships
of 143 human channels
Major branches define
groups of related channel
genes present in the
human genome:
• K+ channels
• TRP channels
• Na+/Ca2+ channels
• CNG and HCN channels
TRP: transient receptor potential channel (eg pain/temperature)
CNG: cyclic nucleotide–gated channel (eg vision & olfaction)
HCN: hyperpolarization-activated channel (rhythmic pacemaker cells)
 Structure of voltage-gated K+ channels (Kv)

Hydrophobicity peaks
correspond to
transmembrane
segments S1-S6 - a
conserved structural
feature of all voltage-
gated K+ channels

S1-S4 segments: voltage sensor domain - S4 contains +vely charged

arginine or lysine residues
S5-P-S6 segments: pore domain - P region contains selectivity filter and
binding sites for toxins and blocking molecules (eg TEA)
 Voltage-gated K+ channel (Kv)

K+ channels are the largest and most diverse family of voltage-gated ion
channels. Humans have at least 79 distinct genes encoding K+ channels.
Kv believed to represent an evolutionary precursor to Nav and Cav
channels because their pore-forming subunit contains only one S1-S6
domain.
Kv channels made up of 4 α subunits & 4 cytoplasmic β subunits.
 Crystal structure of mammalian K+ channel
Kv1.2 (MacKinnon et al 2005)

Four α subunits of the channel, each in a different colour showing the S1-S4 voltage-
sensing domain is separate from the S5-P-S6 pore domain
A. S1-S4 domain of a monomer lies closest to S5-P-S6 domain of adjacent monomer
B. Shows transmembrane domain (TM), intracellular T1 domain allows access of
K + ions to the pore, β subunits coloured according to the α subunit it contacts
 Movement of S4 segment during gating

At rest: +ve charged arginine residues balanced by a neighbouring -ve charge.

Depolarization: +ve charge moved outward until balanced by –ve charge on
adjacent helix, thus stabilizing channel in a new conformation. This screw-type
movement causes the four S6 helices, which form the inner lining of the pore, to
bend away from the pore axis, thereby opening the channel.
 Crystal structure of Streptomyces
K+ channel (KcsA) (MacKinnon et al 1998)

• 2 membrane-spanning elements - analogous to S5-P-S6 portion of

Shaker-type K+ channels (ie missing S1-S4 voltage sensing region)
• P region forms selectivity filter - contains amino-acid residues critical
for K+ selectivity
• 3 K+ ions in conduction pathway
Selectivity Filter
The pore is shaped like an inverted teepee with
narrow selectivity filter ~12 Å long* – minimizes
the distance that K+ interacts with the channel.
Large water-filled cavity and –vely charged helix
dipoles help overcome the electrostatic energy
barrier in the low dielectric membrane center.
Selectivity filter lined by carbonyl oxygen atoms,
providing closely spaced sites with a geometry
favourable for a dehydrated K+ ion but too large
to be energetically favourable for Na+ ion.
Two K+ ions at close proximity in the selectivity
filter repel each other overcoming the otherwise
strong interaction between ion and protein -
allows rapid conduction while maintaining high
selectivity.
* 1 Angstrom = 1 x 10-10 m
 Different classification of K+ channels /currents

K+ currents classified according to functional properties & gating behaviour:

1. Delayed outward rectifiers
2. Transient outward rectifiers (A-type currents)
3. Ca2+ -activated K+ currents
4. Inward rectifiers

These fundamental K+ currents are the manifestation of 5 distinct families

of genes:
1. Kv channels (voltage-gated K+ channels)
2. Small- and intermediate-conductance KCa channels (SKCa and IKCa Ca2+ -
calmodulin–activated K + channels)
3. Large-conductance KCa channels (Ca2+-activated BKCa channels & related
Na+ - and H+- activated K+ channels)
4. Kir channels (inward-rectifier K+ channels)
5. K2P channels (two-pore K+ channels)
Kv (or Shaker-related) channels mediate the delayed
outward-rectifier current and transient A-type current

Delayed outward
rectifier.
• current activates with a
sigmoidal lag phase
(ie is delayed)
• outward current rises
steeply at +ve voltages
(ie is an outward rectifier)
Transient A-type K+ current
 activated and inactivated over a relatively rapid time scale
 activated in the negative Vm range that prevails during the
afterhyperpolarizing phase of action potentials
 important in determining the interval between successive
spikes and thus the timing of repetitive action potentials.
 Kir K+ channels mediate inward-rectifier
K+ currents

• Has only 2 membrane-spanning segments corresponding to S5-P-S6 domain of the

Kv channels (do not contain S1 to S4 segments).
• Conduct K+ current in the inward direction but not in the outward direction when
Mg2+ is present.
• Cytosolic Mg2+ occludes the channel pore and prevents the exit of intracellular K+
• Helps clamp the resting membrane potential close to EK+ and prevents excessive
loss of intracellular K+ during repetitive activity and long-duration action potentials.
 Voltage-gated Na+ channels (Nav)

Inactivation gate

• α subunit (~200 kDa) - channel-forming protein, mediates ionic selectivity

for Na+ , voltage-dependent gating, and sensitivity to neurotoxins (eg TTX).
• β subunits (33 – 38 kDa) - single transmembrane span, accessory role in
modulating channel gating or channel expression.
• 4 domains each containing 6 transmembrane segments (S1-S6)
• S1-S4 = voltage sensing domain
• S5-S6+P-loop = channel pore
• Inactivation gate– S6 domain 3 to S1 domain 4
 Nav Channel α Subunits

CHANNEL HUMAN GENE TISSUE SENSITIVITY TO

PROTEIN TTX (MOLAR)
−9
Nav1.1 SCN1A CNS, PNS, heart 10
−9
Nav1.2 SCN2A CNS 10
−9
Nav1.3 SCN3A CNS, PNS, heart 10
−9
Nav1.4 SCN4A Skeletal muscle, heart 10
−6
Nav1.5 SCN5A Heart, denervated skeletal Insensitive, 10
muscle
−9
Nav1.6 SCN8A CNS, PNS 10
−9
Nav1.7 SCN9A PNS (nociception) 10
−6
Nav1.8 SCN10A PNS (nociception) Insensitive, 10
−6
Nav1.9 SCN11A PNS (nociception) Insensitive, 10
Nax SCN7A CNS/circumventricular organs Unknown
+
(Na sensor)
Nav channel mutations in human genetic
diseases
 Voltage gated Ca2+ channels (Cav)

Consists of α1 subunit (190 kDa) with domains I -IV that form the pore which contain
ionic selectivity, voltage sensitivity, binding sites for various inhibitory drugs etc.
Additional 4 accessory proteins: extracellular α2 subunit (170 kDa), a cytoplasmic β
subunit (55 kDa), and membrane-spanning γ and δ subunits (33 kDa). - accessory
subunits may modulate the gating activity and pharmacology of channel.
Blocked by verapamil, nifedipine etc
 Classification of Cav’s
CHANNEL TYPE
PROPERTY L T N P/Q R
Kinetics Long duration Transient Intermediate to long Intermediate to long Intermediate duration
duration duration
Voltage High threshold Low threshold High threshold High threshold High threshold
activation (> −30 mV) (< −30 mV) (> −30 mV) (> −30 mV) (> −30 mV)
Pharmacology Blocked by DHPs Less sensitive to Insensitive to DHPs, Insensitive to DHPs, Insensitive to DHPs,
DHPs blocked by ω- blocked by ω- ω-conotoxin GVIA,
conotoxin GVIA agatoxin IVA and ω-agatoxin IVA

Location Heart, skeletal Sinoatrial node of Presynaptic Cerebellar Purkinje's Cerebellar granule
muscle, neurons, heart, brain neurons terminals, dendrites, and granule cells, cells, neurons
vascular smooth and cell bodies of cell bodies of central
muscle, uterus, neurons neurons
neuroendocrine cells

Function EC coupling in Repetitive firing of Exocytotic Exocytotic Exocytotic

skeletal muscle, link action potentials in neurotransmitter neurotransmitter neurotransmitter
membrane heart and many release release release
depolarization to neurons
intracellular Ca
signalling
Channel Cav1.1 (CACNA1S) Cav3.1(CACNA1G) Cav2.2(CACNA1B) Cav2.1(CACNA1A) Cav2.3(CACNA1E)
protein (gene) Cav1.2 (CACNA1C) Cav3.2(CACNA1H)
Cav1.3 (CACNA1D) Cav3.3(CACNA1I)
Cav1.4 (CACNA1F)
L-type v’s T-type Calcium channels

Channel behaviour Sensitivity to Ca 2+ channel blockers

DHP’s like
nitrendipine
selectively block
L-type channels

Bay K8644
enhances Ca2+
currents

Phenylalkylamines
(verapamil) and
benzothiazepines
(diltiazem) also
inhibit L-type
Ca 2+ channels but
bind to different
sites from DHP’s
Signal transduction by Cav channels

Ca2+ entering the cell through Cav

channels serves as the second
messenger of electrical signalling,
initiating many different cellular events
including:
• contraction
• secretion
• synaptic transmission
• enzyme regulation
• protein
phosphorylation/dephosphorylation
• gene transcription

Catterall Cold Spring Harb Perspect Biol 2011;3:a003947

 Lambert-Eaton Syndrome
(severe muscle weakness)

Autoimmune disorder affects transmission at NMJ

• antibodies against Cav channels, particularly N and P/Q types (eg Cav2.2, Cav2.1)
• antibodies bind to the S5–S6 linker region of domain III
• decreased Ca2+ influx = reduced amount of ACh released

Affects:
• mainly proximal limb muscles (difficulty climbing stairs)
• can affect respiratory muscles
• weakness decreases with repeated activity
(accumulation of Ca2+ and Ach)
Treatment:
• decreasing ACh breakdown (eg pyridostigmine)
• increasing calcium influx (3,4-diaminopyridine)
• immuno-suppression (eg prednisolone)
 Myotonia

Normal Myotonia
(Slow or delayed relaxation after contraction)

FORCE

EMG

Difficulty releasing grip on tools

Symptoms are worse in cold and sometimes after vigorous exercise
Google “Fainting goats” or “the myotonic goat”
 Myotonia: an ion channel disease
Myotonia congenita - mutations in the CLCN1 gene – Affects 1 in 100 000

Reduced chloride conductance = cumulative after-potential = repetitive firing of AP’s

Normal muscle Myotonia WT Cl--free

Lower threshold for AP generation

Repetitive action potentials and afterpotentials

Compr Physiol. 2015 April ; 5(2): 761–790. doi:10.1002/cphy.c140062.

Channelopathies of Skeletal Muscle Excitability

PAM: Potassium aggravated

myotonia – slower inactivation
causes burst of myotonic
discharges after stimulation
stops

HyperPP: hyperkalemic
periodic paralysis – large
persistent Na2+ current causing
in-excitability and paralysis
Summary
• Voltage gated ion channels typically contain 6 transmembrane
spanning segments. Segments S1-S4 contain the voltage sensor
domain (S4) and segments S5-P-S6 form the pore domain containing
the selectivity filter.
• Membrane depolarization causes movement of S4 opening the
channel and allowing selective diffusion of a specific ion through the
pore.
• Different types of ion currents can be classified according to functional
properties & gating behaviour
• Inactivation gate of Nav channels is formed by the loop between the S6
segment of Domain 3 and the S1 segment of domain 4.
• Mutations in genes encoding for voltage gated ion channels are
responsible for a diverse spectrum of human genetic diseases that are
characterised by the selectivity of the channel for a specific ion and the
tissue in which the channels are expressed.
References: Boron & Boulpaep. Medical Physiology. Chapter 6