principles of expression and functional characterization of mutant ion channels in idiopathic...
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 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