Presentation on theme: "Principles of expression and functional characterization of mutant ion channels in idiopathic epilepsies: Potassium and Calcium Channelopathies Dimitri."— Presentation transcript:
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 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
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
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
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