Principles of expression and functional characterization of mutant ion channels in idiopathic epilepsies:

Potassium and Calcium Channelopathies

Dimitri Kullmann

Institute of Neurology UCL

Action potential

Animation from NEUROBIOLOGY  Molecules, Cells and SystemsGary G. Matthews

Monogenic epilepsies

Type Gene Protein Disease

Voltage-gated channels

Na+ channel

SCN1A subunit of NaV1.1 Generalised epilepsy with febrile seizures plus (GEFS+)

SCN2A subunit of NaV1.2 GEFS+, Benign familial neonatal-infantile seizures

SCN1B 1 subunit GEFS+

K+ KCNQ2 M current Benign familial neonatal convulsions (BFNC), BFNC+myokymia, benign familial infantile convulsions

KCNQ3

Cl- CLN2 ClC-2 Idiopathic generalised epilepsy

Ligand-

Gated channels

Nicotinic ACh receptors

CHRNA2 4 subunit AD nocturnal frontal lobe epilepsy

CHRNB4 2 subunit

GABAA

receptors

GABRG2 2 subunit GEFS+

GABRA1 1 subunit Juvenile myoclonic epilepsy

Not channels EAR domain proteins

LGI1 Epitempin autosomal dominant partial epilepsy with auditory features

MASS1 VLGR1 febrile and afebrile seizures

Other genetic evidence implicating ion channel mutations in epilepsy

Type Gene Protein Disease

Voltage-gated channels

Na+ SCN1A subunit of NaV1.1 Severe myoclonic epilepsy of infancy (SMEI)

K+ KCNA1 subunit of KV1.1 Episodic ataxia type 1 with epilepsy

Ca2+ CACNA1A 1 subunit of CaV2.1

(P/Q-type channel)

Episodic ataxia type 2 with spike-wave seizures

CACNA1H 1 subunit of CaV3.2

(T-type channel)

Childhood absence epilepsy

CACNB4 4 Ca2+ channel subunit Juvenile myoclonic epilepsy

Mutant mice Gene Protein Phenotype

Knockout KCNA1 subunit of KV1.1 Epilepsy, ‘shivering’

CACNA1A 1 subunit of CaV2.1

(P/Q-type channel)

Ataxia, behavioural arrest, spike-wave EEG

Spontaneous mutations: Ca2+ channels

CACNA1A (tottering, leaner, etc)

1 subunit of CaV2.1

CACNB4 (lethargic) 4 subunit

CACNA2D2 (ducky) subunit

K+ channel with accessory subunits

K+ channel with accessory subunits: schematic

Side-view of the core of a K+ channel in the lipid bilayer

Sansom lab. Oxford Univ.

K+ channel selectivity

K+ channel gating

MacKinnon lab, Rockefeller Univ.

K+

translation assembly

targeting

kinetics

permeation

What can go wrong?

Expression models

• cRNA or cDNA injection into Xenopus oocytes

• Transfection of mammalian cell culture

Methods

• Electrophysiology

• Pharmacology

• Immunocytochemistry

• Fluorescence imaging of tagged proteins

Voltage clamp

Two electrode voltage clamp

One electrode voltage clamp(patch clamp)

Voltage-sensingelectrode

Current-passingelectrode

Patch pipette

Oocyte recording

Patch clamp

Cell-attached recordings

Depolarisation: step pipette to negative potentials

-70 mV

0 mV

K+

-70 mV

Cell-attached recordings

Dove et al, 1998

Inward currents (downward deflections) are currents going from pipette into cell

e.g. Ca2+ channels

-70 mV

-70 mVCa2+

Whole-cell recordings

-70 mV

-70 mV

K+

0 mV

K+

K+

0 mV

Depolarisation: step pipette to 0

Inward currents are going from bath into cell

KCNQ2 and KCNQ3 co-assemble to form heterotetramers

Wang et al (1998)

KCNQ2-3 hetorotetramers underlie Im

Jentsch, 2000

Biervert et al (1998)

KCNQ2 mutation in BFNC causes decreased IK

25% reduction in IM current is sufficient to cause disease (Schroeder et al, 1998)

BNFC mutations

(BNFC + myokymia)

Truncations

Dedek et al, 2001

Voltage sensor mutations affect activation kinetics

Ca2+ channelopathies

Gene Channel Disease

Muscle CACNA1S subunit of CaV1.1 HypoK periodic paralysisMalignant hyperthermia

RYR1 Ryanodine receptor (sarcoplasmic channel)

Malignant hyperthermiaCentral core disease

Neuronal CACNA1A 1 subunit of CaV2.1

(P/Q-type channel)

Familial hemiplegic migraineEpisodic ataxia type 2Spinocerebellar ataxia type 6Absence epilepsy?

CACNA1H 1 subunit of CaV3.2

(T-type channel)

Childhood absence epilepsy

4 subunit mutations also reported in association with epilepsy/episodic ataxia

Ca2+ channel structure

2

1

Ca2+ channel classification

Expressed in thalamus

Crunelli lab. Cardiff

Experimental absence seizure

Perez-Reyes, 2000

T-type Ca2+ channels contribute to burst firing

CaV3.2 mutations affect activation and inactivation kinetics

Khosravani et al, 2004Predicts gain of function

Conclusions

• Mutations can have multiple consequences for ion channel function

• Channelopathies must be seen in the context of neuronal and circuit function

• K+ channels stabilise or repolarise membranes

• Loss-of-function KCNQ2 and KCNQ3 mutations are associated with epilepsy

• Ca2+ channels have multiple roles in transmitter release, signal transduction and electrical properties of neurons

• T-type channels contribute to burst-firing of thalamic neurons

• Gain-of-function mutations have been found in a few sporadic cases of childhood absence epilepsy