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 Systems Gary G. Matthews

Monogenic epilepsies TypeGeneProteinDisease Voltage-gated channels Na + channel SCN1A  subunit of Na V 1.1 Generalised epilepsy with febrile seizures plus (GEFS+) SCN2A  subunit of Na V 1.2 GEFS+, Benign familial neonatal- infantile seizures SCN1B  1 subunit GEFS+ K+K+ KCNQ2M currentBenign familial neonatal convulsions (BFNC), BFNC+myokymia, benign familial infantile convulsions KCNQ3 Cl - CLN2ClC-2Idiopathic generalised epilepsy Ligand- Gated channels Nicotinic ACh receptors CHRNA2  4 subunit AD nocturnal frontal lobe epilepsy CHRNB4  2 subunit GABA A receptors GABRG2  2 subunit GEFS+ GABRA1  1 subunit Juvenile myoclonic epilepsy Not channelsEAR domain proteins LGI1Epitempinautosomal dominant partial epilepsy with auditory features MASS1VLGR1febrile and afebrile seizures

Other genetic evidence implicating ion channel mutations in epilepsy TypeGeneProteinDisease Voltage-gated channels Na + SCN1A  subunit of Na V 1.1 Severe myoclonic epilepsy of infancy (SMEI) K+K+ KCNA1  subunit of K V 1.1 Episodic ataxia type 1 with epilepsy Ca 2+ CACNA1A  1 subunit of Ca V 2.1 (P/Q-type channel) Episodic ataxia type 2 with spike- wave seizures CACNA1H  1 subunit of Ca V 3.2 (T-type channel) Childhood absence epilepsy CACNB4  4 Ca 2+ channel subunit Juvenile myoclonic epilepsy Mutant miceGeneProteinPhenotype KnockoutKCNA1  subunit of K V 1.1 Epilepsy, ‘shivering’ CACNA1A  1 subunit of Ca V 2.1 (P/Q-type channel) Ataxia, behavioural arrest, spike-wave EEG Spontaneous mutations: Ca 2+ channels CACNA1A (tottering, leaner, etc)  1 subunit of Ca V 2.1 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+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-sensing electrode Current-passing electrode Patch pipette

Oocyte recording

Patch clamp

Cell-attached recordings Depolarisation: step pipette to negative potentials -70 mV 0 mV K+K+ -70 mV

Cell-attached recordings Dove et al, 1998 Inward currents (downward deflections) are currents going from pipette into cell e.g. Ca 2+ channels -70 mV Ca 2+

Whole-cell recordings -70 mV K+K+ 0 mV K+K+ K+K+ 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 I m Jentsch, 2000

Biervert et al (1998) KCNQ2 mutation in BFNC causes decreased I K 25% reduction in I M 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

Ca 2+ channelopathies GeneChannelDisease MuscleCACNA1S  subunit of Ca V 1.1 HypoK periodic paralysis Malignant hyperthermia RYR1Ryanodine receptor (sarcoplasmic channel) Malignant hyperthermia Central core disease NeuronalCACNA1A  1 subunit of Ca V 2.1 (P/Q-type channel) Familial hemiplegic migraine Episodic ataxia type 2 Spinocerebellar ataxia type 6 Absence epilepsy? CACNA1H  1 subunit of Ca V 3.2 (T-type channel) Childhood absence epilepsy  4 subunit mutations also reported in association with epilepsy/episodic ataxia

Ca 2+ channel structure   22 11

Ca 2+ channel classification Expressed in thalamus

Crunelli lab. Cardiff Experimental absence seizure

Perez-Reyes, 2000 T-type Ca 2+ channels contribute to burst firing

Ca V 3.2 mutations affect activation and inactivation kinetics Khosravani et al, 2004 Predicts 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 Ca 2+ 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