Etude des canaux ioniques: intrts pour la physiopathologie et le traitement des troubles de la motricit

Cours international: mdecine gnomique, du diagnostic la thrapie 17-21 octobre 2016-Institut Pasteur de Tunis

Arnaud Monteil

arnaud.monteil@igf.cnrs.fr

Ion channels

- Ion channels are gated pores that permit the passive flow of ions down their electrochemical gradients.

- More than 400 genes are known that encode ion channel subunits. - Alternative splicing and heteromeric assembly of different subunits increase the diversity of ion

channels. - Such many channels are needed to accomplish very complex cellular functions. - Dysfunction of ion channels are key events in many pathological processes. - Ion channels are target of importance in a pathological context.

Ion channel classes

Ashcroft, 2006

Some examples of currents

Piezo2 (mechano-gated) Nav1.3 (voltage-gated)

nAChR (Ligand-gated, direct) NALCN (Ligand-gated through GPCRs)

3D models depicting VGNCs in 3 different states

Kim, 2014

Introduction

Introduction

Skeletal muscle channelopathies

1- Mutations in AchR subunits causes myasthenia (muscle weakness) by preventing binding of acetylcholine. 2- Loss of presynaptic K+-channel function (KV1.1, KCNA1) leads to increased transmitter release and enhanced muscle contraction. 3- Downregulation of presynaptic Ca2+ channels causes myasthenia by preventing neurotransmitter release. 4- Gain-of-function mutations in the muscle Na+ channel (Nav1.4, SCN4A) cause hyperexcitability and myotonia. 5- Loss-of-function mutations in ClC channels cause hyperexcitability and myotonia. 6- Loss-of- function mutations in Kir1.1 cause hyperexcitability and myotonia. 7- Mutations in muscle CaV channels (Cav1.1, CACNA1S) impair Ca

2+ release from intracellular stores, producing malignant hyperthermia or paralysis. 8- RYR channels impair Ca2+ release from intracellular stores, producing malignant hyperthermia or paralysis.

Ashcroft, 2006

Skeletal muscle channelopathies

Kim, 2014

Skeletal muscle channelopathies

Cannon, 2015

Recurrent episodes of weakness, lasting minutes to hours, with spontaneous full recovery.

Provocation of attacks by environmental stresses: - Rest after a period of vigorous exercise.

- Carbohydrate-rich meals.

- Shifts of serum potassium (high or low).

- Exposure to cold.

- Emotional stress.

- Pregnancy.

Over times, some patients develop a slowly permanent weakness.

Periodic Paralysis

The transient loss of muscle excitability during an attack of weakness is caused by depolarization of the resting membrane potential.

Three different mechanisms have been identified. - A persistent Na+ caused by a defect of inactivation (1-2% remains open), other defects in

gain-of-function mutants (gating, inactivation, hyperpolarized shift of activation).

- Loss-of-function changes for inward rectifier potassium channels (Kir2.1, Kir2.6, Kir3.4).

- Gating pore current.

Periodic Paralysis

Rudel et al, 1984

Hypokalemic Periodic Paralysis (CACNA1S, SCN4A)

Hyperkalemic Periodic Paralysis (SCN4A)

Hyperkalemic Periodic Paralysis (SCN4A)

Hyperkalemic Periodic Paralysis (SCN4A)

Therapeutic management of periodic paralysis

Acetazolamide is beneficial for about 50% of HypoPP patients with Cav1.1 but not Nav1.4 mutations.

K+-supplements (HypoPP).

Avoidance of large carbohydrate-rich meals (HypoPP).

KATP openers (cromakalin; HypoPP).

Na+-K+-Cl- co-transporter (NKCC) inhibitor (bumetanide; HypoPP).

Avoidance of K+-rich food (Nav1.4 - HyperPP).

Carbohydrate snack to truncate an episode (Nav1.4 - HyperPP).

Promote kaliuresis with diuretics (Nav1.4 - HyperPP).

Carbonic anhydrase inhibitors (acetazolamide, dichlorphenamide; Nav1.4 - HyperPP).

Inability of muscle to relax after voluntary effort.

The after-contractions may persist for many seconds.

With repeated movements, the intensity of myotonia diminishes over seconds to minutes and may even become asymptomatic.

Conversely, some affected individuals have paradoxical worsening of myotonic stiffness with repeated effort (paramyotonia). This process is aggravated by muscle cooling.

Additional triggers have been associated with worsering myotonia: - Potassium administration.

- Emotional stress.

- Pregnancy.

- Hypothyroidism.

- Depolarizing general anesthetics.

- Cold exposure.

Myotonia

Myotonia

Two different mechanisms have been identified: - Reduction of the resting chloride conductance.

- Gain-of-function changes to the voltage-dependent gating of Nav1.4 sodium channels.

Myotonia congenita (CLCN-1)

- Divided into dominant (Thomsen disease) and recessive (Becker disease) forms. - Over 200 mutations in the CLCN1 gene have been reported.

some cases can be total, a direct treatment tar-geted to the CLC-1 protein is practically impos-sible. T heoretically, direct treatment could bepossible if the function is only partially reducedas in the case of dominant myotonia or in thecase of mutations that have residual function as,e.g., the M485V mutation that reduces thesingle-channel conductance [Wollnik et al.,1997]. A drug that increases the open probabil-ity could then increase the Cl conductance ofthe skeletal muscle and abolish the hyperexcit-ability. T he pharmacological agents that inter-fere most strongly with CLC-1 are 9-anthracenecarboxylic acid (9AC) and the S() enantiomerof p-chloro-phenoxy-propionic acid (CPP), bothof which inhibit the muscle Cl conductance andCLC-1with an apparent affinity in the 10-50 mMrange [Palade and Barchi, 1977; De Luca et al.,1992; Steinmeyer et al., 1991b; Aromataris etal., 1999; Pusch et al., 2000]. CPP and also 9ACare most likely open channel blockers that, inaddition, reduce the open probability by imped-

ing channel opening of closed, drug-bound chan-nels [Pusch et al., 2001; Accardi and Pusch, un-published observations]. These substances aretherefore not suited to increase CLC-1 mutantactivity.

A pharmacological treatment could also beaimed at increasing non-specifically the Cl con-ductance of muscle. No useful drug with thatproperty is, however, known so far.

In practice, many patients manage the situa-tion without any medication. If treatment is nec-essary, in general the muscle fibers are renderedless excitable by partially inhibiting the voltage-gated Na+ channel with local anesthetics suchas mexiletine or related drugs [Lehmann-Hornand Jurkat-Rott, 1999].

Dominant and recessive myotonia are causedby mutations of the same gene coding for themuscle Cl channel, CLC-1. T he mechanismbehind the different modes of inheritance of

FIGURE 2. Localization of missense mutations. The approximate position of recessive (blue squares), dominant (redcircles), semi-dominant (red-blue hexagons), and sporadic (green squares) mutations is shown in a topology model of theprimary protein sequence. The length of the various segments is approximately to scale. The topology was chosen ac-cording to Schmidt-Rose and Jentsch [1997].

Pusch, 2002

Popponen et al, 2008

Myotonia congenita (recessive mutations of CLCN-1)

Kubisch et al, 1998

Myotonia congenita (dominant mutations of CLCN-1)

Kubisch et al, 1998

Myotonia congenita (SCN4A)

Paramyotonia congenita (SCN4A)

Avoidance of cold environments.

Voltage-gated sodium channels blockers (tocainimide, mexitetine, flecainimide).

Acetazolamide (carbonic anhydrase inhibitor)?

Therapeutic management of Myotonia congenita

Disease ChannelProtein Gene

Cognitive impairment with orwithoutcerebellarataxia

Nav1.6: sodium channel, voltage-gated, type VIII, subunit

SCN8A

Episodicataxiatype1

Kv1.1:potassiumchannel,voltage-gated,shaker-relatedsubfamily,member1

KCNA1

Episodicataxiatype2

Cav2.1:calciumchannel,voltage-gated,P/Qtype,1Asubunit

CACNA1A

Episodicataxiatype5 Cav4:calciumchannel,voltage-gated,4subunit CACNB4

Spinocerebellarataxiatype6

Cav2.1:calciumchannel,voltage-gated,P/Qtype,1Asubunit

CACNA1A

Spinocerebellarataxiatype13

Kv3.3: potassium channel, voltage-gated, Shaw-relatedsubfamily,member3

KCNC3

Autosomal-Dominant CerebellarAtaxia

Cav3.1: calcium channel, voltage-gated, T type, 1Gsubunit

CACNA1G

CLIFAHDDsyndrome(dominant) NALCN:sodiumchannel,leak,subunit NALCN

Infantile hypotonia withpsychomotor retardation and

characteristic facies (IHPRF,recessive)

NALCN:sodiumchannel,leak,subunit NALCN

Channelopathies-related ataxia

Snutch & Monteil, Neuron. 2007 May 24;54(4):505-7.

Cladogram of Subunits for the 4-Domain Ion Channel Family

- Central Nervous System - Heart - Adrenal Gland - Thyroid Gland - Salivary Gland - Mammary Gland - Islets of Langerhans

Dominant-Negative Effects of Misfolded Mutants of VGCC

Mezghrani et al, 2008

NALCN regulates the neuronal resting membrane potential

Hippocampal neurons M. musculus

(Lu et al, 2007, Cell)

RPeD1 neurons L. stagnalis

(Lu et al, 2011, Plos ONE)

Premotor interneurons C. elegans

(Xie et al, 2013, Neuron)

Retrotrapezoid nucleus neurons M. musculus

(Shi et al, 2016, J. Neurosci.)

C4 nerve root recordings from brain stem spinal cord M. musculus

(Lu et al, 2007, Cell)

Congenital contractures of limbs and face, hypotonia and developmental delay (Chong et al, 2015, Am J Hum Genet; Aoyagi et al, 2015, Hum Mutat; Wang et al, 2016, Clin Genet; Fukai et al, 2016, J Hum Genet; Karakaya et al, 2016, Neuropediatrics; Sivaraman et al, 2016, J Clin Neurosci; Bend et al, 2016, Neurology)

Infantile neuroaxonal dystrophy (Kroglu et al, 2013, J Med Genet)

Infantile hypotonia with psychomotor retardation and characteristic facies (Al-Sayed et al, 2013, Am J Hum Genet ; Gal et al, 2016, Eur J Med Genet)

NALCN in human diseases

Dominant-negative effect of NALCN mutations (CLIFFAHDD)

Chong et al, 2015

Aoyagi et al, 2015

An animal model for the CLIFFAHDD syndrome

Correction of NALCN deficiency by acting on other channels

Kasap et al, 2016

Correction of NALCN deficiency by acting on other channels

(4-AP: inhibitor of voltage-gated K+ channels)

Correction of NALCN deficiency by acting on other channels

(Quinine: inhibitor of voltage-gated K+ channels)

Correction of NALCN deficiency by acting on other channels

Correction of NALCN deficiency by acting on other channels

(FPL-64176: activator of voltage-gated Ca2+ channels)

Correction of NALCN deficiency by acting on other channels

(CBNX: activator of Gap Junctions)

Correction of NALCN deficiency by acting on other channels

(MFQ: activator of Gap Junctions)

Akinsie: L'akinsie est une lenteur d'initiation des mouvements avec une tendance l'immobilit (mouvements volontaires, mouvements associs, mouvements d'ajustement postural, mouvements d'expression gestuelle et motionnelle), et ce, en l'absence de paralysie. Cela est d un problme d'activation de zones du cerveau (atteinte de la voie nigro-strie entranant un dficit en dopamine).

Dyskinsie: la dyskinsie activit motrice involontaire, lente et strotype affectant prfrentiellement la face (langue, lvres, mchoire) stendant au tronc et aux membres.

Dysplasie: Une dysplasie est une malformation ou dformation rsultant d'une anomalie du dveloppement d'un tissu ou d'un organe, qui survient au cours de la priode embryonnaire ou aprs la naissance.

Myotonie: une myotonie se caractrise par une dcontraction lente et difficile d'un muscle la suite d'une contraction volontaire.

Paralysie priodique: Les paralysies priodiques sont un groupe de maladies gntiques rares qui conduisent une faiblesse musculaire ou une paralysie (rarement la mort) partir de facteurs dclenchant courants tels que le froid, la chaleur, des repas riches en glucides, le jene, le stress, l'excitation et l'activit physique de toute nature.

Ataxie: l'ataxie est une pathologie neuromusculaire qui consiste en un manque de coordination fine des mouvements volontaires. Elle n'est pas lie une dficience physique des muscles mais plutt une atteinte du systme nerveux. Le trouble de la coordination est partiellement corrig par le contrle visuel.

Dfinitions