Periodic paralysis and voltage-gated ion channels

Kidney International, i/ol, 49 (1996), pp. 9—18

EDITORIAL REVIEW

Periodic paralysis and voltage-gated ion channels mary structure of the main subunits of the sodium, calcium and potassium channels demonstrated that they share similar structural organizations, and are members of a family which probably evolved from a common ancestral gene. A recently identified Escherichia coil homologue of an eukaryotic potassium channel [7] indicates that the structure has been conserved for least 2 billion years, antidating the divergence between prokaryotes and eukaryotes. The structure of these channels consists of four homologous transmembrane domains, each composed of six transmembrane a helices (Si to S6) (Fig. 1A). The amino- and carboxy-termini and the linker regions between domains are all located in the cytoplasm. Each domain of the potassium channel is encoded by a

Periodic paralyses are a group of muscle disorders sharing a common clinical feature consisting of episodic attacks of muscle

weakness due to sarcolemmal membrane depolarization. This type of muscle weakness may be induced by modifications in blood

potassium levels related to renal or intestinal deficits. The occurrence of muscle weakness secondary to changes in blood potas-

sium levels is rare, however, and is only observed in cases of profound hypokalemia. In contrast, attacks of muscle weakness dominate the clinical picture in primary periodic paralyses. Primary periodic paralyses are familial disorders which were separated into hypo- and hyperkalemic types, on the basis of the observed modifications in blood potassium levels during the attacks. Recent advances in the understanding of the pathophys-

separate gene so that the functional channel is a homo- or

iology of primary periodic paralyses have shown that both hypoand hyperkalemic periodic paralysis are caused by mutations in

hetero-tetramer. The four domains of the sodium and calcium channels are encoded by a single gene [reviewed in 8]. The ion channel comprises an intramembrane pore in the center of a channel, respectively. square array defined by the homologous domains. Each domain The family of voltage-gated ion channels contributes to the wall of the pore (Fig. 1B) [reviewed in 9]. The Electrical excitability of muscle involves changes in sodium and fourth transmembrane segment in each domain (S4) has a major potassium permeabilities. Under physiological conditions, synap- effect on the voltage-dependence of activation. It contains re-

voltage-gated ion channels, the muscle calcium and sodium

tic transmission between nerve and muscle triggers local depolar- peated rings composed of positively-charged amino acids followed ization at the endplate, leading to the firing of action potentials. by two hydrophobic residues. This sequence is thought to form an During the rising phase of the action potential, voltage-gated amphiphatic helix exposing positively-charged residues within the sodium channels activate rapidly, allowing an influx of sodium and membrane which act as voltage-sensors [10].

thus, membrane depolarization. Before the peak of the action potential, sodium channels start to inactivate, and the membrane permeability to sodium drops. At the same time, a delayed and

Voltage-gated calcium channels

At least five types of voltage-gated calcium channels (termed L, slow activation of voltage-gated potassium channels takes place, T, N, P and 0) have been distinguished by their electrophysiologiinitiating an outward current which hastens membrane repolarcal and pharmacological properties [reviewed in 2, 11]. For ization. By a regenerative process, the action potential propagates example, L-type channels (L for long lasting) can be blocked by along the sarcolemma, invading the depth of the transverse the calcium channel antagonists 1,4-dihydropyridines, phenylalkytubular system, where it activates voltage-gated calcium channels lamines and benzothiazepines, but they are generally insensitive involved in the excitation-contraction coupling process. to the peptide w-conotoxin. Voltage-gated ion channels are protein complexes composed of The composition of the calcium channel was first obtained by a principal subunit (the a-subunit) that forms the ion pore by biochemical purification of L-type channels from transverse tubuitself, and of auxiliary subunits which influence activation and lar membranes of rabbit skeletal muscle using dihydropyridines inactivation [reviewed in 1—3]. These channels are expressed in a [121. The channel consists of four distinct subunits: al (175 kD), variety of cells. Their subunit composition and their structure a2Th (175 kD), /3(52 kD) and y (32 kD). The components of the depend on the cell type. The sodium channel from skeletal muscle channel complex associate specifically, as assessed by co-immucontains a large a-subunit (260 kD) and a non-covalently associ- noprecipitation with monospecific antibodies and by co-sedimenated /3-subunit of approximately 38 kD, similar to the brain tation in sucrose gradients, with a stoechiometric ratio of 1:1:1:1 /31-subunit [41. The skeletal calcium channel consists of an for each subunit [reviewed in 5, 6]. All subunits of the skeletal al-subunit associated with transmembrane a2I- and y-subunits dihydropyridine L-type calcium channel were cloned and seand an intracellular /3-subunit [5, 6]. [13—17]. Purification, molecular cloning and determination of the pri- quenced The al-subunit is in itself sufficient to form a functional calcium channel as demonstrated by direct expression in Xenopus oocytes and in mammalian L cells [18—21]. The contribution of the other subunits of the calcium channel Received for publication April 3, 1995 was determined by co-expression of the al-subunit with either /3-, and in revised form August 7, 1995

a2/- and y-subunits in Xenopus oocytes or mammalian cells (CHO, HEK293 or L cells) [reviewed in 1, 2, 22, 23]. The

Accepted for publication August 7, 1995

© 1996 by the International Society of Nephrology

9

10

Fontaine et at: Periodic paralysis

A

Domain II

Domain I

Domain IV

Domain III

/

/

Argi 239His Argi 239Gly

A rg528 H is

B

IONS

Membrane

NH2

COOH

Fig. 1. L-type calcium channel al-subunit structure. (A) Schematic representation of the al-subunit, and localization of the two predominant hypoPP mutations. The al-subunit consists of four homologous repeats (domain Ito IV) containing six membrane spanning a-helices (Si to S6). The position of the SS1 and SS2 segments implicated in ion selectivity of the channel are also shown. The positions of the two main mutations (arg528his, argl239his or arg1239 gly) found in hypoPP families are also shown. These mutations are located in the fourth segment of the second and the fourth domain, respectively. (B) Schematic representation of the tertiary structure of the al-subunit. Each homologous repeat (domain Ito IV) is located within the membrane surrounding a central transmembrane pore.

magnitude of the calcium current expressed in these cellular autosomal recessive mutation blocks the expression of the alsystems using the al-subunit was enhanced by co-expression of subunit of the calcium channel, eliminating the L-type calcium the a2/b-subunit. The kinetic properties of the channel and of the current, reducing intramembrane charge movement and suppressdihydropyridine binding site were also altered [18, 20, 24, 25]. ing excitation-contraction coupling (E-C coupling) necessary for Co-expression of the J3- and of the al-subunits increased the the release of calcium from the sarcoplasmic reticulum [reviewed calcium current directed by the al-subunit several-fold. The in 31, 32]. Expression of the skeletal muscle al-subunit in mutant 13-subunit also accelerated both the activation and inactivation cells rescues the mdg phenotype [33]. kinetics of the calcium current [20, 24—291. Moreover, co-expresTo identify the regions which determine the properties of the sion of the a2Ib- and 13- subunits to form a al/a21b113 complex, al-subunit, chimeric subunits were constructed combining segdramatically enhanced the peak current, indicating considerable ments of skeletal and cardiac muscle al-subunits, and analyzed for current waveform, calcium dependence of E-C coupling, and synergism [20, 24]. A mammalian system which expresses only the auxiliary sub- intramembrane charge movement. The region implicated in E-C units, the muscular dysgenesis (mdg) mouse mutant, was used to coupling was identified as the large intracellular loop which define the structural elements of the al-subunit necessary for connects domains II and III [34]. The first domain (DI) contribchannel function in muscle [reviewed in 2, 23, 30]. This fatal utes to the activation kinetic of the calcium channel [35]. Chimeric

Fontaine et al: Periodic paralysis

11

amino acid sequence rich in lysines and arginines, hut with 4 prolines, allowing it to form a highly flexible charged element. isoform, showed that the S3 segment and the linker connecting S3 This is the shortest segment linking any of the repeat domains, and S4 in repeat I were responsible for the difference in activation suggesting that domains III and IV may be physically coupled kinetics between cardiac and skeletal muscle L-type calcium [reviewed in 43] [44]. first domains (DI) constructed by substituting each transmembrane segment (Si to S6) of the skeletal domain with the cardiac

channels [36]. The molecular determinants of ion selectivity were also identified. In sodium and potassium channels, the SS1-SS2 region of the

Complementary DNA clones encoding the /31-subunit of the rat

brain and skeletal muscle sodium channels were isolated. The deduced primary structures indicated that the /31-subunit was a

S5-S6 linker region has been postulated to line the channel pore protein of 23 kD containing a small cytoplasmic domain, a single and to play a major role in ion selectivity. In the calcium channel, transmembrane segment and a large extracellular domain [1]. the S52 segment has also been implicated in ion selectivity, since Expression of cloned a-subunits in Xenopus laevis oocytes substitution of glutamate residues for lysinc in the SS2 segments yielded functional sodium channels [46, 47]. Co-injection into

of the S5-S6 linker from regions I (DI) and III (DIII) altered

oocytes of mRNAs encoding domains I to III and mRNAs

encoding only the domain IV of the a-subunit suggested that all segment of the DIII and DIV domains transformed a sodium four domains were required to form functional sodium channels selectivity [37, 38]. Even a single amino acid change within the SS2

channel into a calcium channel [39].

even when linkage between domains was interrupted [48]. InacTo identify the region of the calcium channel implicated in tivation was slower, however, in expressed a-subunits than in dihydropyridine binding, chimeric calcium channels were con- neurons or muscle, and the voltage-dependence of inactivation structed with al-subunits from L-type channels from cardiac was shifted to more positive membrane potentials. Co-expression muscle, sensitive to dihydropyridines, and from brain, insensitive of the a- and f31-subunits from rat brain or skeletal muscle in to dihydropyridines, and were expressed in Xenopus oocytes. The oocytes accelerated inactivation, and shifted its voltage-depeneffects of dihydropyridine agonists and antagonists on these dence to more negative membrane potentials [1]. The TFX chimeric proteins demonstrated that the linker between S5 and S6 binding site located near the outer entrance of the channel in domain IV (DIV) of the L-type cardiac calcium channel was a blocked ion flow either by physically occluding the channel or by major site of dihydropyridine action [40]. inducing a conformational change in the protein [45]. The role of the S4 helix as a voltage-sensor was first evaluated The mechanism of inactivation of calcium channels has also been extensively studied. The SS2-S6 region of domain III (DIII) by introducing point mutations into the S4 segment converting is important for inactivation [40]. Other studies showed that the positively charged amino acid residues into neutral or negativelyamino acids responsible for the kinetics of inactivation were charged residues [48]. A reduction in positive charges in segment located in the membrane-spanning segment S6 of the first repeat S4 of domain I decreased the slope of the voltage-dependence of (DI), and the extracellular and cytoplasmic region flanking S6 activation. Inactivation of sodium channels is not strongly voltage-depen[41]. The /3-subunits also play an important role in the inactivation kinetics of calcium channels [reviewed in 1, 2, 22, 23]. A conserved dent. The apparent voltage-dependence of inactivation is due to

motif between repeats I and II which binds the /3-subunit was

the coupling of inactivation to voltage-dependent activation.

recently identified [42]. Mutations within this motif (approximate-

Studies using site-directed antibodies and site-directed mutagenesis in the interdomain III-IV indicate that this intracellular loop

ly 20 amino acids) reduced stimulation of peak currents by the /3-subunit and altered the inactivation kinetics and the voltagedependence of activation.

is involved in inactivation [48, 49]. Slowed inactivation was

observed but effects on the voltage-dependence of the activation or on the steady-state properties of inactivation were unremarkVoltage-gated sodium channels able. Using alanine-scanning mutagenesis, it was suggested that Pharmacological studies have demonstrated that mammalian residues near the intracellular end of segment S6 of domain IV skeletal muscle expresses two types of sodium channels distin- play a role in fast sodium channel inactivation [50].

guished by their sensitivity to tetrodotoxin (TTX): the TTXand the TTX-insensitive sodium channel which is expressed in

Hypokalemic periodic paralysis (hypoPP) Familial periodic paralysis was recognized as a clinical entity as

embryonic and denervated muscle, as well as in the heart [43—45].

early as the end of the nineteenth century. A decrease in blood

The protein components of sodium channels were first identi-

potassium levels during attacks of periodic paralysis was described in 1934 by Biemond and Daniels [51], and founded the basis for the first classification of periodic paralyses. A review of the earlier literature concerning hypokalemic periodic paralysis (hypoPP) is in [52—54]. The prevalence of hypoPP has been estimated to be

sensitive sodium channel which is the adult isoform of the protein,

fled by photoaffinity labeling with derivatives of a-scorpion toxins.

To determine the primary structure of sodium channels, sodium channel a-subunit cDNAs from mammalian brain and muscle, as

well as from eel and Drosophila were cloned and sequenced [reviewed in 8, 10]. All known sodium channels share a common

molecular structure. The primary sequence consists of 1800 to 2000 amino acids (260 kD) defining four large repeat domains which exhibit internal homology at the amino acid level. Each of these domains contain six regions capable of forming transmem-

brane helices designated Si to S6. There is more sequence

1/100,000. The disease is transmitted as an autosomal dominant trait with reduced penetrance and expression in women. Women

may even escape expression of the phenotype, although they transmit the disease. In consequence, a male to female predominance of 3:1 has been reported [52]. We recently re-evaluated the

penetrance of hypoPP and found it to be 80% in women and

homology among the repeat domains within a given species than 100% in men, in the families studied [55]. As in other dominant between species, and low homology among interdomain regions, diseases, new mutations have also been reported as well as except for interdomain III-IV. Interdomain Ill-TV contains a 53 "sporadic" cases where there was no documented evidence of

12

Fontaine et a!: Periodic paralysis

transmission to the descendance. The sporadic cases reported were more frequently men than women. In light of the recent findings, since the early cases lacked a molecular analysis, one may

suppose that some of these cases were actually familial, with asymptomatic women in the genealogy, as we recently reported [56].

The age of onset of hypoPP is within the second decade of life:

muscles and replacement of the muscles by fat [61]. The origin of the vacuoles has been debated. Since the work of Engel [58], the vacuoles are considered to evolve from the sarcoplasmic reticulum and the tubular system as the result of proliferation and degeneration of these membranous organelles. Consequently, there is communication with the extracellular space as demonstrated by the entry of extracellular peroxydase dye into the vacuoles [58].

60% of patients develop the disease before the age of 16 [52], The absence of correlations between the number and the although younger or later onsets have been reported. severity of attacks and the vacuolar myopathy, as well as the lack The main provocative factors are rest after exercice and meals of vacuoles at the onset of the disease, or in muscle fibers rich in carbohydrates. Other factors include emotion, stress, cold rendered inexcitable [581, suggests that periodic paralysis attacks exposure and alcohol ingestion. Typical attacks occur at night and and the vacuolar myopathy are two different phenotypes associthe patient awakes paralyzed. The paralysis is flaccid, involving ated with an identical primary gene defect. Attacks represent a the four limbs. Deglutition and ocular muscles are usually spared, highly penetrant phenotype contrary to the vacuolar myopathy as is the diaphragm. The intercostal muscles may be affected, which is only observed in a third of the patients [58]. The factors, however, resulting in difficult coughing and expectorating. Mild genetic or epigenetic, triggering and controlling the expression of attacks limited to the lower limbs or only to a group of muscles, these phenotypes are unknown. Other morphological changes are also occur. If attacks develop upon rest after exercice, they may be

also observed in periodic paralysis. Tubular aggregates, which are

aborted by pursuing a mild effort. Concomitant to the attack, a decrease in blood potassium levels is observed due to movement of potassium from the extracellular compartment into muscles. There is no direct involvement of the heart, but modifications of the electrocardiogram, directly related to the decrease in blood

proliferations of the lateral sacs on longitudinal components of the sarcoplasmic reticulum, are localized under the sarcolemma. Other changes include an increase in central nuclei, abnormal variation in the fiber size, fiber necrosis and proliferation of connective tissue elements. Symptomatic treatment of hypoPP attacks by oral administration of potassium salts is usually sufficient. Severe attacks with a

potassium levels are observed. Blood potassium levels may fall to

1 mmol/liter. When the diagnosis is difficult, an attack may be provoked by infusion of 2 g/kg of glucose and 0.1 unit/kg of profound hypokalemia may require intravenous infusion of potasinsulin. This test may induce profound hypokalemia and a severe sium salts under strict medical supervision in an intensive care attack, and it should therefore be performed only under strict unit. Preventive measures include avoidance of the provocative factors, and oral administration of potassium salts and acetazolThe frequency of attacks is variable. They are usually more amide [62]. frequent in the second and third decade, and decline in frequency HypoPP is a voltage-gated muscle calcium channel disorder as the patient gets older. Attacks may occur daily resulting in an medical supervision.

apparently permanent but reversible state of muscle weakness. An attack may last from a few hours to a few days. Some patients may

Electrophysiological studies performed in vitro on muscle fibers from hypoPP patients revealed alterations in membrane excitabil-

only present a few attacks over their lifetime, or even a single ity. An unexpected observation concerned the reduction in the attack. Contrary to hyperPP, myotonic manifestations are never resting membrane potential. Hypokalemia should, indeed, induce observed, either clinically or by electromyography. Patients with a membrane hyperpolarization instead of the observed depolarizalid-lag sign have been reported [57], but this sign is not specific to tion. An increase in the permeability ratio NaIK was also myotonia and may be observed in normal individuals. reported, and was mainly attributed to an elevated sodium

Independent of the severity and the frequency of periodic conductance (GN). The increase in GNa was not suppressed by paralysis attacks, and even in the absence of attacks, patients may specific sodium channel blockers excluding, therefore, this ion develop permanent muscle weakness due to vacuolar myopathy channel as the primary target in hypoPP [631. Nevertheless, these [reviewed in 58]. This permanent muscle weakness should be observations pointed out to ion channels as possible clues in the distinguished from subintrant attacks causing a prolonged but pathophysiology of the disease. reversible state of muscle weakness. The onset of the vacuolar It was possible to profit from the existence of large families myopathy is usually in the fourth or fifth decade of life but cases affected by the disease and to use linkage studies to localize the occurring as early as in childhood have been reported [59]. The hypoPP gene. Given the electrophysiological data, the strategy same morphological changes are evidenced in both hyper- and chosen for identifying the hypoPP gene was to localize the hypoPP hypokalemic periodic paralyses. They consist of a vacuolar myopathy first described by Goldflam in 1895 [60]. The vacuoles are not

specific to periodic paralysis and may be seen in the end-plate myopathy of the slow-channel syndrome, and even in other muscle disorders such as myotubular myopathy or polymyositis. Vacuoles in muscle are now considered to reflect the myopathic process in periodic paralysis even if occasional observations of vacuoles in

gene on the human genome, and to look for a known or an unknown ion channel in the region [64, 65]. The initial linkage studies were conducted with three families of Portuguese, German and French descent. Positive lod-scores were

observed for markers localized on chromosome 1q31 to 32, establishing the hypoPP-1 locus [55]. The calcium channel alsubunit CACNL1A3, also called the dihydropyridine receptor (DHP receptor), mapped to the same region in the same 5 CM

patients without a permanent muscle weakness, or cases with a permanent muscle weakness and no vacuoles, have been reported interval as the hypoPP-1 locus [55]. Mutations were subsequently [reviewed in 58]. Vacuolar myopathy affects mostly the muscles of found in the coding sequence of CACNL1A3, establishing it as the pelvic girdle and the proximal muscles of upper and lower the hypoPP gene [66, 67]. Three mutations affecting two amino limbs. CT-scans demonstrate hypodense cores of the affected acids contained within segment S4 were demonstrated. One

Fontaine et al: Periodic paralysis

replaces a positively charged arginine by a weakly-positive histidine in position 528 in segment S4 of domain II (Fig. 1A). The other two mutations replace an arginine by either a histidine or a glycine in position 1239 in segment S4 of domain IV (Fig. 1A). The two arginines affected by the hypoPP mutations belong to the ring of positively-charged amino acids composing segment S4. The role of these arginines is crucial, since it is conserved in the primary structure of CACNL1A3 among different species and in

13

role is, however, more difficult to understand than in hyperPP (see

below). Exercise and meals rich in carbohydrates result in high blood levels of adrenalin and insulin, both known to stimulate sodium-potassium ATPase activity [71]. High pump activity may,

in turn, lead to hypokalemia. The pump is electrogenic, and makes a slight contribution to the resting membrane potential. However, the potential Ep, brought by the pump is directly related to membrane resistance by Ohm law. Hence, when the

other members of the voltage-gated ion channel family. The membrane is depolarized, its resistance drops, and Ep becomes arg528his and argl239his mutations were found in a large number of hypoPP families [66, 67]. In a series of 16 hypoPP families of Caucasian origin, 8 presented the arg528his amino acid change, and the other 8, the argl239his mutation [56]. The phenotype was identical in terms of attacks and of the development of a vacuolar myopathy. However, incomplete penetrance was observed only in women with the arg528his mutation [561. Screening of these two CACNL1A3 main mutations is therefore of diagnostic value in hypoPP. Because of the incomplete penetrance of the arg528his

negligible. A difficult task is to understand how hypokalemia interacts with

lotypes segregating with the mutations demonstrated that the predominance of these two mutations was not due to a founder

membranous formations coupling the T-tubular system to the

effect, but to recurrent mutation. One hypoPP family, which was also linked to chromosome 1q31 to 32, did not show either of the two arginine mutations (unpublished data), suggesting that other

the T-tubular system and the sarcoplasmic reticulum. Their

the mutated channel to lead to muscle depolarization and paralysis. The link between channel mutation and muscle disorders may involve changes in the expression and/or function of other ionic channels and cellular processes contributing to membrane excitability.

Expression of the mutated channel in muscle cells might also give insight into the processes underlying vacuolar myopathy. In mutation, genotyping of all at-risk individuals, and especially the mouse mdg mutant, the absence of expression of the dihydrowomen, should be performed [56]. Characterization of the hap- pyridine receptor results in disorganization of the triads which are

mutations in the calcium channel CACNLIA3 remain to be discovered. Furthermore, although mutations in the CACNL1A3

gene are a major cause of hypoPP, at least two families with hypoPP were not genetically linked to chromosome 1q31 to 32. HypoPP is therefore genetically heterogeneous [681.

The amino acid changes observed in hypoPP result in an autosomal dominant phenotype which suggest that both the normal and the mutated allele are expressed. This is in contrast with the mdg mutation which concerns the coding sequence of the DHP receptor causing a shift in the translational reading frame [69]. Consequently, no expression of the DHP receptor is observed in skeletal muscles of homozygous mice. The mdg phenotype consists in neonatal death from respiratory arrest due to complete paralysis of the skeletal muscle. The skeletal muscle DHP receptor acts both as the L-type calcium channel and as the voltage sensor for E-C coupling, but does not play a key role in membrane excitation. Hence, genetic

linkage of hypoPP to the a-i subunit of the DHP receptor is rather puzzling. Recent analyses of the L-type calcium currents, recorded from

sarcoplasmic reticulum. As described above, vacuoles arise from occurrence could be investigated by following the formation of the

T-tubular system and the sarcoplasmic reticulum in developing muscle cells transfected with the mutated calcium channel gene. Thyrotoxic hypoPP is another type of hypoPP. Unlike familial hypoPP, thyrotoxic hypoPP occurs sporadically in 95% of cases. Thyrotoxic hypoPP is 70 times more common in males than in females, and affects Orientals in 75% of cases. In hyperthyroid Chinese, the risk of developing the disease is 15 to 20%. Clinical manifestations are similar to familial hypoPP and cease when the euthyroid state is restored. The dihydropyridine receptor, may also account for the pathophysiology of thyrotoxic hypoPP. Thyroid hormones stimulate the sodium-potassium pump ATPase, and could, thereby, induce hypokalemia. Susceptible persons in the Oriental population might carry a dihydropyridine receptor

allele, insufficient by itself to confer hypoPP, but able, in a

hyperthyroid state, to cause thyrotoxic hypoPP. This hypothesis is theoretically testable by characterizing dihydropyridine receptor alleles in a population with thyrotoxic hypoPP.

Hyperkalemic periodic paralysis (hyperPP) The complete description of Hyperkalemic periodic paralysis

(hyperPP) is credited to Gamstorp [72], but Tyler and his

collaborators also reported a similar family [73]. HyperPP is a muscle disorder of genetic origin transmitted with an autosomal mode of inheritance. The penetrance is high, close to 100%. Sporadic cases and neo-mutations also occur, although the majority of patients belong to families displaying the trait. The prevalence of the disease is not known. Patients with hyperPP either mutation [70]. These findings do not provide a direct clue to the mechanism present acute attacks of muscle weakness accompanied by an underlying muscle paralysis, nor do they explain the abnormal increase in blood potassium levels. The age of onset is usually in behavior of the sarcolemmal membrane of hypoPP patients. the first years of life, before the patient begins to walk. The

myotubes obtained from muscle biopsies of hypoPP patients, revealed a shift in the steady state inactivation curve toward negative potentials in the arg528his mutation, and a decrease in the current density in the argl239his mutation. No alteration of the voltage-dependent activation of the channel was found in

frequency of attacks is variable, ranging from one every month to tion are currently undertaken. In addition, studies on the inter- several per week. The duration of attacks is also variable, from 10 action of the mutated al-subunit with the other subunits of the minutes to two hours. Attacks most often affect the lower limbs, calcium channel are also underway, using in vitro expression and sometimes the upper limbs. The muscles of the head and neck Therefore, more thorough analyses of the DHP receptor dysfunc-

systems.

Triggering factors are necessary to produce an attack. Their

are usually spared as well as the diaphragm. Besides these classical manifestations, attacks may be atypical by the length of duration

14

Fontaine et al: Periodic paralysis Table

Phenotype

1.

Mutation

Mutations in the muscle sodium channel and their corresponding phenotype Substitution

Channel part

References

Abnormal inactivation Shift in the voltage-dependence of activation Abnormal inactivation

[86, 101, 102]

HyperPP NormoPP

C2188T

HyperPP PC HyperPP HyperPP SCM SCM (acetazo lamideresponsive)

G3466A

DI1IS4/S5

Alall56Thr

A4078G A4774G

DIVS1 DIVS6

Metl36OVal Metl592Val

nd Abnormal inactivation

[87, 102]

Ser804Met Ilell6OVal

nd nd

[88] [95]

Abnormal inactivation Abnormal inactivation Abnormal inactivation Abnormal inactivation Abnormal inactivation Abnormal inactivation Abnormal inactivation Abnormal inactivation

[89, 921 [92] [92] [89, 103] [93, 103] [90, 103, 104] [90, 103, 104] [91, 105]

PC/SCM SCM SCM PC/SCM

PC PC PC SCM

DIIS5

Electrophysiology

Thr7O4Met

C2411T

DIIS6

A3555G

DIIIS4/S5

G3917T G3917A G3917C C3938T T4298G C4342T C4343A G4765A

Dill-DIV Dill-DIV Dill-DIV Dill-DIV

Glyl3O6Val Glyl3O6Glu Glyi3O6Ala

DIVS3 DIVS4 DIVS4 DIVS6

Leul433Arg Argl448Cys

Thrl3l3Met Argl448flis Vall589Met

[88, 103] [94]

DI to DIV and Si to S6 indicate the domains and the segments of the sodium channel, respectively. Abbreviations are: HyperPP, hyperkalemic periodic paralysis; NormoPP, normokalemic periodic paralysis; PC, paramyotonia congenita; SCM, sodium channel myotonia. Original references are cited.

(several days to months), their location in a single limb, the involvement of head and neck muscles or the diaphragm. Attacks occur when resting after exercice, and may be aborted by resum-

ing the exercise. Attacks may be provoked by a meal low in

of autosomal dominant inheritance [781. Paramyotonia was differentiated from myotonia congenita which is now known to be a chloride channel disorder [79, 80]. Contrary to myotonia congenita, where a warm-up phenomenon is observed, the myotonia

carbohydrates or by cooling the muscles. Myotonic manifestations are frequently observed in hyperPP

in paramyotonia is aggravated by exercise. Furthermore, PC

patients. Myotonia is caused by a prolonged failure of muscle decontraction. It is characterized by high-frequency repetitive

Symptoms may also be provoked by ingestion of potassium salts as

discharges of sarcolemmal membrane producing a "dive-bomber" sound on the audio-monitor during recording by electromyography. "True" myotonia is ameliorated by repetitive movements, the so-called "warm-up phenomena," whereas paramyotonia or "paradoxical myotonia" worsens during exercice. Contraty to the attacks,

patients present muscle stiffness and weakness induced by cold.

observed in hyperPP. However, contrary to hyperPP, vacuolar myopathy is never observed in PC. In hyperPP, successful prevention is most frequently obtained by frequent meals of high carbohydrate content and avoidance of fasting, exposure to cold, or overexertion. If these precautions are not sufficient, diuretic agents such as acetazolamide or thiazides

myotonic manifestations may be more constant and therefore of are usually effective in the prevention of attacks of muscle importance for the diagnosis. The patient may present cramps or weakness. muscle stiffness during exercice or when exposed to cold. The HyperPP is a voltage-gated muscle sodium channel disorder most frequent clinical myotonic manifestation is in the eyelids. Percussion myotonia is usually not encountered. In hyperPP The clue to identify the hyperPP gene was reported by Lehpatients with the same known genetic defect, clinical manifesta- mann-Horn and collaborators [81], who observed the occurrence

tions of myotonia were present in 25% of cases, and typical of non-inactivating sodium currents in muscle fibers from hyperPP electromyographic signs in 50% of cases [74]. A diagnosis of patients, when the extracellular potassium concentration was hyperPP should, therefore, be considered in a patient presenting periodic paralysis with myotonia, and the diagnosis of hypoPP regarded as unlikely. The absence of myotonia does not, however, exclude the diagnosis. Hyperkalemia is usually mild and does not induce cardiac dysrhythmia. In patients with the same known genetic defect, hyperkalemia was only demonstrated in 50% of patients even when repeated measurements of blood potassium levels were performed during the attacks [741. Normokalemic periodic paralysis and hyperPP are in fact the same clinical and genetic entity. Later in life, patients develop a vacuolar myopathy which cannot be distinguished from that observed in hypoPP [75]. Clinical variants of hyperPP have been described. The most notable syndrome is hyperPP with paramyotonia [76, 771. Cosegregation of both disorders in same families led to the hypothesis that hyperPP and paramyotonia congenita (PC) were allelic disorders. PC was described last century as a myotonic syndrome

increased. These results provided strong arguments for the implication of abnormal sodium channels in hyperPP [811. Following the characterization of the rodent muscle sodium channel gene [44], the human equivalent was isolated and a polymorphism was

found, enabling linkage analysis [82]. Co-segregation of the sodium channel was first observed in a large family with hyperPP, suggesting that the primary defect resided in the sodium channel gene [82]. Similar observations were made in hyperPP families of other ethnic backgrounds, as well as in PC families, demonstrating that hyperPP and PC were allelic disorders [83—85]. Mutations

were found in the coding sequence of the sodium channel gene establishing it as the hyperPP gene [86, 87]. To date, fourteen point mutations have been found in the human sodium channel gene [86—951. Table 1 summarizes these mutations and the genotype/phenotype correlations. Two of the mutations causing hyperPP, Thr7O4Met and Metl592Val [74, 95, 96], account for

15

Fontaine et al: Periodic paralysis

approximately 75% and 25% of the hyperPP families studied, normal sodium channels, thereby impairing the main key for

ing in the Ravensberg area of Germany [98]. Genetic and

membrane excitation. Periods of hyperexeitability and myotonia could be explained by the fine balance between the extent of membrane depolarization determined by the opening of mutated sodium channels, and the efficiency of membrane repolarization due to potassium outfiux. Large depolarizations would lead inevitably to membrane inexeitability. Limited depolarization could still allow normal sodium channels to reactivate and support, for limited periods, repetitive action potentials.

molecular analysis of the sodium channel permitted re-evaluation of families displaying autosomal dominant myotonic syndromes

the paralysis attack may be explained by an increase in the

respectively [74, 95, 961. No founder effect could be demonstrated by characterization of the haplotypes segregating with mutations [74, 96]. As shown in Table 1, the variety of mutations is greater in PC than in hyperPP [93]. The Thrl3l3Met mutation was found

in a majority of French families [74]. No founder effect was demonstrable, however, arguing in favor of recurrent mutations in the sodium channel gene [74, 97]. A founder effect for PC could only be demonstrated for an unusual cluster of families originat-

resembling myotonia congenita caused by a chloride channel defect. A myotonic syndrome either termed "acetazolamideresponsive myotonia congenita" or "sodium channel myotonia (SCM)" has therefore been added to the list of sodium channel

The return to the resting membrane potential and the end of sodium-potassium ATPase activity. This effect would be triggered by the accumulated changes in the sodium and potassium gradients. Consistent with a restoration in the ion balance, a polyurie

HyperPP also affects Quarter horses. The mutation has been disseminated by selective breeding among approximately 5% of

crisis is often observed at the end of an attack. High sodium-potassium pump activity may also intervene for preventing the attacks. According to Lehmann-Horn and collaborators [54], interruption of paralysis attacks by mild exereiee may

the Quarter horse population [100]. Genetic linkage to the

be due to the stimulation of the sodium-potassium pump by

muscle disorders [95, 99].

sodium channel gene has been demonstrated, and a point muta- adrenaline and acidosis. The attacks will then only occur after rest tion was found that changes amino acid 1410 from a phenylala- [71]. For the same reason, the protective effect of acetazolamide nine to a leucine located in segment 3 of domain IV of the sodium might also be due to the induction of long-term acidosis. Fasting is also a provocative factor in hyperPP. It is known to decrease channel [100]. To investigate the dysfunction of mutated channels, in vitro insulin secretion, thereby diminishing the protective effect of high expression studies were conducted. The Thr7O4Met and the sodium-potassium ATPase activity.

Metl592Val mutations were introduced into the rat skeletal muscle channel which was then expressed in a human embryonic kidney cell line [101, 102]. Both mutations disrupted inactivation without affecting single channel conductance. The Thr7O4Met mutation caused late first opening, prolonged open times and a

negative shift in the voltage-dependence of activation [101]. Neither of the mutant channels were sensitive to increases in extraeellular potassium. Cummins and collaborators thought that the hyperpolarizing shift in the voltage-dependence of activation was the most important effect [101], while Cannon and Strittmatter put an emphasis on the magnitude of the sodium influx [102]. Mutations identified in PC and SCM patients have been introduced either in rat or human skeletal muscle sodium channel gene and expressed in different cell types. The mutants ArgI 448Cys, Argll48His, AlallS6Thr, Thrl3l3Met, Leul433Arg, Glyl3O6AJa, Glyl3O6Glu, Glyl3O6Val and Vall589Met similarly disturbed channel inactivation, and altered voltage-dependence [91, 102— 105]. Neither the Argl448Cys, Argl448His, Ala1l56Thr, Thr1313

M or Leul433Arg mutants affected the activation. Analysis of

single channel recordings revealed that Argl448Cys and Argl448His mutant channels inactivated normally from closed

states, but poorly from the open state [104]. How do these observations compare with the in vivo situation? Single channel recordings from muscle biopsies of patients clearly demonstrated abnormal inactivation of the sodium channel in hyperPP [106,

107]. In contrast to normal muscle, the proportion of noninactivating channels increased to 3 to 5% in high potassium

Conclusion

In the past few years, periodic paralyses were shown to be due to mutations in muscle voltage-gated ion channels. Hypokalemie and hyperkalemie periodic paralysis are diseases of calcium and

sodium channels, respectively. These mutations are not lethal, causing only subtle effects on channel behavior. Analysis of the dysfunction of these natural mutations, which affect regions not previously investigated by in vitro mutagenesis, has already increased our knowledge on the mechanism of sodium channel inactivation. The understanding of the complete disease phenotypes is, however, far from complete. The bases of the modifications of blood potassium levels and of the vaeuolar myopathy are still unknown. The mechanism underlying vacuolar myopathy might be elucidated by studies of the effect of mutated channels on the development and the physiology of the tubulo-reticular system in muscle. Co-expression studies with mutated channels, other channel subunits or other ion channels should help to understand the disease phenotypes as well as the interactions between ion channels. They will also help to develop effective drug therapies. BERTRAND FONTAINE, PA5CALE LAPIE, EMMANUELLE PLA55ART, NACIRA

TABTI, SOPHIE NICOlE, JOcELYNE REBOUL, and CLAIRE-SOPHIE RIME-DAvOINE

Fédération de Neurologie and INSERM U134, Hopital de Ia Salpêtrière, Paris, and Departement de Physiologie Générale, Université Paris XII, Créteil, France

concentration [106, 107].

How might a periodic paralysis attack occur? An increase in extraeellular potassium induces membrane depolarization. Even a slight depolarization may be sufficient to activate mutated chan-

nels characterized by low activation voltage threshold. Noninactivating sodium current will develop and lead to sustained membrane depolarization. This will result in the inactivation of

Acknowledgments This work was supported by grants from INSERM, AFM, GREG and AP-HP. Reprint requests to Bertrand Fontaine, M.D., JNSERM U134, Hopital de la Salpetrière, 47 boulevard de l'Hopital, 75013 Paris, France.

16

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