Selected examples on

The Structure of Ion Channels

Ion channels Classification by gating Voltage-gated channels Voltage-gated Na+, Ca2+ , K+ channels

Classification by selectivity Na+ channels Ca2+ channels

Transient receptor potential (TRP) channels

K+ channels

Cyclic nucleotide-gated channels

H+ channels

Ligand-gated channels Ionotropic receptors ATP-sensitive channels

Cl- channels Anion channels Cation channels

Cyclic nucleotide-gated channels

Inward rectifiers Light-gated channels Mechanosensitive channels

Gap junctions

Ligand-gated ion channels Cys-loop receptors GABAA receptors

Anion selective

Glycin receptors nACh receptors Serotonin Receptors

Ionotropic glutamate receptors Hyperpolarization activated cyclic nucleotide-gated channels ATP-sensitive potassium channels

Cation selective

Ligand-gated ion channels → Cys-loop receptors → general structure

Ligand-gated ion channels → Cys-loop receptors → general structure

5-fold rotational symmetry

Ligand-gated ion channels → Cys-loop receptors → Ligand-gated ion channels – Cys-loop receptors General structure Extracellular General structure –→ Extracellular domaindomain

Ligand binding site

Allosteric modulator binding site

Ligand binding site

Ligand-gated ion channels → Cys-loop receptors → Ligand-gated ion channels – Cys-loop receptors General structure Extracellular General structure –→ Extracellular domaindomain • the „binding pocket” is formed by one principal and one complementary subunit • Binding pocket residues: • A, B, C loops from the principal subunit • D, E, F β-sheets from the complementary subunit • 2 bindings sites are required for channel activation • ECD contracts around agonists, relaxes around antagonists • cation-π interactions

Ligand-gated ion channels → Cys-loop receptors → General structure → Transmembrane domain

M1: may be involved in transmitting movements from ligand-bound ECD to M2 M2-M3 loop: critical role in transmitting the energy of binding into channel opening M2: provides pore-lining residues, constitutes the gate M3 and M4: shield M2 from the lipid environment

Ligand-gated ion channels → Cys-loop receptors → General structure → Transmembrane domain

Hydrophobic girdle

Ion selectivity filter

hydrophobic charged

Anionic channels (GABAA, Gly-R)

Cationic channels (nACh-R, 5-HT-R )

-1` position: Glutamic acid

-1` position: uncharged

Ligand-gated ion channels → Cys-loop receptors → GABAA receptors → Subunits Class

IUPHAR protein name

Gene

α1

GABRA1

α2

GABRA2

α3

GABRA3

α4

GABRA4

α5

GABRA5

α6

GABRA6

β1

GABRB1

β2

GABRB2

β3

GABRB3

γ1

GABRG1

γ2

GABRG2

γ3

GABRG3

delta

δ

GABRD

epsilon

ε

GABRE

pi

π

GABRP

theta

θ

GABRQ

ρ1

GABRR1

ρ2

GABRR2

ρ3

GABRR3

alpha

beta

gamma

rho

GABA is the most common neurotransmitter in the CNS GABAA receptors found in 40% of synapses in the brain GABA-R classes: GABAA: ionotropic GABAB: G-protein-coupled There are 19 different GABA-R receptor subunits

Most abundant subunit classes: alpha, beta, gamma, delta (rho is expressed in the retina) GABAA is anion selective

Ligand-gated ion channels → Cys-loop receptors → GABAA receptors → Subunit stoichiometry two different α and two different β subunits can be present ϒ cannot be precipitated with other ϒsubunits δ is not present with ϒ 2 α, 2 β and 1 ϒ or δ subunits make up the channel estimated number of different subtypes: 800

•α1 - β2 - ϒ2 is the most common composition α2βγ2, α3βγ2, α4βγ2, α5βγ2, α6βγ2, α4βδ and α6βδ are also common to lower extent

a

Ligand-gated ion channels → Cys-loop receptors → GABAA receptors → Allosteric modulaMon

BZ1 class: contain the α1 subunit cortex, talamus, cerebellum sedative, amnesia

BZ2 class: contain the α2 subunit limbic system, motor neurons anxiolytic, myorelaxant

Ligand-gated ion channels → Cys-loop receptors → Glycine receptors Gly is one of the most common neurotransmitter in the CNS Gly-R is anion selective 5 known subunits: α 1 – 4 and β Developmental switch of isoforms: fetal form α2 homomers adult form: α1β heteromers Subunit stoichiometry: 3α:2β long-held dogma, no evidence

Ligand-gated ion channels → Cys-loop receptors → Nicotinic Acetylcholine Receptors Cys-loop receptor signature structure

Ligand-gated ion channels → Cys-loop receptors → Nicotinic Acetylcholine Receptors nACh-R is a cation channel

17 known subunits: α 1 – 10 β1 – 5 δ ϒ ε

Na+ (inward) and K+ (outward) ions flow through the open channel, net current is inward (depolarizing) Muscle type nAChR (neuromuscular junction): α1, β1, γ, and δ – embryonic α1, β1, δ, and ε - adult 2:1:1:1 ratio

Neural type nAChR (central or peripheral nervous system): homo- or heteromeric combinations of α2−α10 and β2−β4 subunits e.g. (α4)3(β2)2, (α4)2(β2)3, (α7)5

Ligand-gated ion channels → Cys-loop receptors → Ionotropic Serotonin Receptors Serotonin: 5-hydroxitryptamine

5-HT receptors

Family Type Mechanism 5-HT1 Gi/Go-protein coupled. Decreasing cellular levels of cAMP. 5-HT2 Gq/G11-protein coupled. Increasing cellular levels of IP3 and DAG. 5-HT3 Ligand-gated Na+ and K+ cation channel. Depolarizing plasma membrane. 5-HT4 Gs-protein coupled. Increasing cellular levels of cAMP. 5-HT5 Gi/Go-protein coupled. Decreasing cellular levels of cAMP. 5-HT6 Gs-protein coupled. Increasing cellular levels of cAMP. 5-HT7 Gs-protein coupled. Increasing cellular levels of cAMP.

5 genes encoding for 5 subunits HTR3A 5-HT3A HTR3B 5-HT3B HTR3C 5-HT3C HTR3D 5-HT3D HTR3E 5-HT3E

Potential Inhibitory Excitatory Excitatory Excitatory Inhibitory Excitatory Excitatory

Ligand-gated ion channels → Glutamate Receptors (no Cys-loops here) Nervous system: Glutamate is the main excitatory neurotransmitter, present in 50% of nervous tissue

Glutamic Acid

Taste buds: GluR is responsible for the transmission of the umami taste stimuli

Clinical conditions:

Autism Attention Deficit Hyperactivity Disorder (ADHD) Diabetes Multiple sclerosis Schizophrenia etc.

Ligand-gated ion channels → Glutamate Receptors

Ionotropic

Metabotropic

AMPA receptors

Kainate receptors

NMDA receptors

GluA1-4

GluK1-5

GluN 9 isoforms

Homo- or heteromers

GluK1-3: homo- or heteromers

(GluN1)2 + (GluN2)2 or GluN1 + GluN2

GluK4-5: “silent subunits”

Four subunits assemble in one channel

mGluR

Ligand-gated ion channels → Glutamate Receptors

ATD: amino-terminal domain

LBD: ligand binding domain TMD: transmembrane domain CTD: carboxy-terminal domain

Ligand-gated ion channels → Glutamate Receptors

Tetrameric structure

Ligand-gated ion channels → Glutamate Receptors Dimer of dimers!

Same polypeptide

Different conformations!

Ligand-gated ion channels → Glutamate Receptors

Ligand-gated ion channels → Glutamate Receptors

Ligand binding-induced conformation changes

Voltage-gated ion channels → General membrane topology

Inward rectifiers Tetrameric structure

Membrane topology of one subunit

Inward rectifiers 2 1 3 0

AP 4

4

Is it an inward current? IK1 “Linear” current-voltage relationship Blockade by cytoplasmic Mg2+ and polyamines

Inward rectifiers and voltage-gated channels → Ion selectivity

Conserved selectivity filter motif

Voltage-gated ion channels → General membrane topology

Voltage-gated ion channels → Conformation transitions

Closed

Open

Closed

Open

Inactivated

N-type inactivation

C-type inactivation

Voltage-gated ion channels → Conformation transitions → Various kinetics contributes to functional diversity Six TMD structure

Transient outward current

Delayed rectifier current

2

2

1

1 3

3 0

0

4 mV 5050mV -35mV mV -35 -80mV mV -80

4

4

50mV

-40mV

4

Voltage-gated ion channels → Auxiliary subunits

The minK story The cardiac delayed rectifier KvLQT1 drives no current in homomeric form

A single TMD protein called minK was injected into Xenopus oocytes → huge delayed rectifier potassium current is generated.

How is that possible?

Voltage-gated ion channels → Auxiliary subunits Molecular toolkit: the KCNE gene family KCNE genes KCNE1 - minK KCNE2 – MiRP1 KCNE3 – MiRP2 KCNE4 – MiRP3 KCNE5 – MiRP4

Voltage-gated ion channels → Auxiliary subunits Molecular toolkit: the KCNE gene family KvLQT1 + minK

KvLQT1 + MiRP2

KvLQT1 + MiRP4

Voltage-gated ion channels → Diversity of potassium channels

Voltage-gated ion channels → Sodium channels “Tetramer”-mimicking structure

2 1 3 0

4

4

All encoded by a single giant polypeptide

Voltage-gated ion channels → Calcium channels

α2δ subunit

β subunit Increased traficking

Increase current amplitude

Left-shifted voltage dependence of activation

Faster activation and inactivation Left-shifted voltage dependence of inactivation