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Recent Patents on Biotechnology, 2012, 6, 184-191

Lessons Learned from Muscle Fatigue: Implications for Treatment of Patients with Hyperkalemic Periodic Paralysis Jean-Marc Renaud1* and Lawrence J. Hayward2 1

University of Ottawa, Department of Cellular and Molecular Medicine, 451 Smyth Rd., Ottawa Ontario, Canada, K1H 8M5; 2Neurology Department, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, USA 01655 Received: June 30, 2012

Revised: July 30, 2012

Accepted: August 04, 2012

Abstract: Hyperkalemic periodic paralysis (HyperKPP) is a disease characterized by periods of myotonic discharges and paralytic attacks causing weakness, the latter associated with increases in plasma [K+]. The myotonic discharge is due to increased Na+ influx through defective Na+ channels that triggers generation of several action potentials. The subsequent increase in extracellular K+ concentration causes excessive membrane depolarization that inactivates Na+ channels triggering the paralysis. None of the available treatments is fully effective. This paper reviews the capacity of Na+ K+ ATPase pumps, KATP and ClC-1 Cl- channels in improving membrane excitability during muscle activity and how using these three membrane components we can study future and more effective treatments for HyperKPP patients. The review of current patents related to HyperKPP reinforces the need of novel approaches for the treatment of this channelopathy.

Keywords: Skeletal muscle, muscle fatigue, muscle paralysis, hyperkalemic periodic paralysis, patients, humans, treatments, pain, muscle soreness, muscle spasms, muscle weakness, potassium, plasma potassium levels, Na+/K+ ATPase pumps, patents. INTRODUCTION Skeletal muscle contracts when stimulated by motoneurons. Upon stimulation, muscles generate action potentials that spread along the surface membrane and t-tubules where they activate the sarcoplasmic reticulum to release Ca2+ for the activation of the contractile components. Action potentials depend on voltage-sensitive Na+ (Nav) and K+ (Kv) channels. In the resting state, the cell membrane potential (EM) is about -80 mV [1]. Nav channels are activated when the cell membrane is depolarized to about -60 mV and their opening gives rise to the action potential depolarization phase due to a large Na+ influx causing EM to reach +30 mV at the peak of the action potential. The repolarization phase, during which EM returns back to -80 mV, depends on two events. First, Nav channels become inactivated, a process known as fast inactivation, which prevents further Na+ influx. Second, Kv channels are activated allowing a large K+ efflux. At rest, Nav and Kv channels are closed and resting EM is maintained by three channels, the ClC-1 Cl- channel, the strong K+ inward rectifier Kir2.1 and the KATP channel [2-7]. Finally, there is a voltage-sensitive Ca2+ channel (CaV1.1), also known as L-type Ca2+ channel and dihydropyridine receptor (DHPR), that is located in t-tubules and acts as the voltage sensor that triggers Ca2+ release from the sarcoplasmic reticulum during an action potential. Mutations in the Cl-, Na+, Ca2+, and K+ channels have been recognized to cause diseases known as channelopathies. In skeletal muscle, channelopathies span a spectrum of *Address correspondence to this author at the University of Ottawa, Department of Cellular and Molecular Medicine, 451 Smyth Rd.Ottawa Ontario, Canada K1H 8M5; Tel: (613) 562-5800, x8156; Fax: (613) 562-5434; E-mail: [email protected] 2212-4012/12 $100.00+.00

electrical disturbances with two extremes with regard to membrane excitability. Myotonia is characterized by an increased membrane excitability resulting in repetitive contractions often leading to stiffness due to a reduced capacity to relax. Periodic paralysis, on the other hand, involves episodic decreases in membrane excitability leading to weakness that typically lasts for several hours, but in some cases can last for days or even weeks [8,9]. Hyperkalemic periodic paralysis (HyperKPP) is among the channelopathies for which symptoms include both myotonia and paralysis. Several studies on membrane excitability have demonstrated that the activity of pumps and channels can be modified under some physiological conditions to maintain membrane excitability when changes in ion concentration reach levels that depress membrane excitability. Under different physiological conditions such as when muscle fibers face a metabolic stress such as fatigue, pump and channel activity is modified to depress membrane excitability in order to reduce contractility so that energy levels can be preserved. The objective here is first to review what is known about HyperKPP and the physiological role and regulation of the Na+ K+ ATPase pump, ClC-1 and KATP channels during muscle activity. Then, using what we know about how muscle fibers used the three membrane components to improve membrane excitability, we will discuss potential targets for the development of better pharmacological strategies to treat patients suffering of HyperKPP. HYPERKALEMIC PERIODIC PARALYSIS Symptoms and Treatments HyperKPP is an autosomal dominant disease with complete or nearly complete penetrance [10,11]. The disease is characterized by periods of myotonic discharges that occur © 2012 Bentham Science Publishers

Muscle Fatigue and Treatment of HyperKPP

during and between paralytic attacks causing weakness [8,9,11-14]. Paralysis is associated with increases in plasma [K+] from the normal 4 mM to 6-8 mM [8,10-12,15]. Affected individuals generally do not complain of muscle stiffness, but myotonic discharges are detected in 75% of cases. Weakness is prominent in the limbs [9,10,14] and may completely incapacitate patients because they are unable to move [11,14]. The duration of the attacks varies between and within patients. Patients experience 1 to 3 attacks per day lasting a few minutes to hours. Some attacks can last a few days, with extreme cases lasting several months [8,9,10,11]. In general, 44% of patients have the first attack prior to the age of five, and 92% before the age of ten [10,13]. The attacks are short and frequent in childhood and become longer and severe during adolescence [9,10-12]. Past the age of 30, while myotonic discharge and paralysis may become much less prominent, patients can suffer from debilitating myopathy such that walking becomes a difficult task. In some cases, patients become wheelchair bound [8,9,10,11], the weakness being primarily due to fiber damage [8,9,11,15]. The most common, potent, and consistent trigger (7080% of the time) for an attack is a period of rest after strenuous exercise [8-15]. Cold exposure, fasting, and emotional stress constitute other less consistent triggers. Ingestion of KCl consistently triggers an attack in all HyperKPP patients but not in normal individuals, suggesting that the former have a greater sensitivity to increased extracellular [K+] ([K+]e) [16,17]. For the purpose of this review, [K+]e will refer to the [K+] when speaking of more than one extracellular compartments in vivo as well as the [K+] in physiological bathing solutions when muscles are tested in vitro. Patients often feel a “creeping heaviness” beginning in the thighs prior to a paralytic attack. An attack can sometimes be avoided by mild exercise such as walking [9,10,12, 13]. This is an interesting paradox because mild exercise causes an increase in [K+]e [18-21] and yet it prevents paralysis under these circumstances. Ingestion of calcium gluconate or acetazolamide are two major pharmacological treatments. Calcium partially alleviates the severity of the attacks [10,11], while acetazolamide, a carbonic anhydrase inhibitor, reduces the frequency but not the severity of attacks [9,11,15,12] but becomes ineffective over time [12]. None of the available treatments is fully effective, and their underlying mechanisms of action are incompletely understood. HyperKPP is Associated with Mutations of the SCN4A Gene Na+ channels are composed of at least two protein subunits known as - and -subunits. The -subunit is the largest, forms the pore of the channel, and contains all of the activation and inactivation components. The protein is composed of four homologous domains (I-IV), each domain containing six trans-membrane spans (S1-S6) [22]. The membrane spans S1-S3 are believed to interact with the phospholipid bilayers of the cell membrane. S4 contains multiple positively charged amino acid residues and is the voltage sensor that activates the channel to open. S5 and S6 surround the pore of the channels with the extracellular hairpin loop between S5 and S6 responsible for the channel specificity to

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Na+. The fast channel inactivation that occurs during an action potential involves three major residues located in the intracellular loop between domains III and IV. The -subunit expressed in skeletal muscle [8,23,24] is the NaV1.4 isoform, which is encoded by the SCN4A gene. It is now well established that HyperKPP is linked to seven missense mutations in the SCN4A gene [25]. This is why other excitable tissues, such as cardiac muscle and neurons, are not affected as they express other Nav isoforms; i.e., cardiac muscles express NaV1.5 while neurons express NaV1.1NaV1.3 and NaV1.6-NaV1.9 [26-28]. Most (~70%) of the HyperKPP cases are related to threonine to methionine (T704M) and methionine to valine (M1592V) mutations [8]. Patients with the T704M mutation usually suffer their first paralytic attack before the age of one yr. and the frequency varies from 8 to 42 attacks per month. In comparison, patients with the M1592V mutation suffer their first attack later (~ 5 yrs. old) and less frequently (5-6 attacks per month). The mean duration of the paralysis is about 10 times longer for the M1592V mutation than for the T704M (89h vs. 8h), with maximum duration as long as 7 days for both mutations [8]. One case has been reported in which a patient with the M1592V mutation has all the HyperKPP symptoms except that plasma [K+] remains normal during paralytic attacks, the authors suggested in their study that this mutation may be considered a variant of HyperKPP and not a distinct disease entity known as normokalemic periodic paralysis [14]. Interestingly, except for one case [29], HyperKPP patients do not suffer from respiratory distress [14] despite the fact that the diaphragm, which is a major respiratory muscle, also expresses Nav1.4 [30]. Effects of Missense Mutations on NaV1.4 Channel and Physiology of Skeletal Muscles More than 30 missense mutations in the SCN4A gene have now been found [31]. Mutations affecting the fast inactivation process usually result in hyperexcitability of the cell membrane, leading to myotonia and potassium-aggravated myotonia [31,32]. Mutations located in the S4 spans lead to Hypokalemic Periodic Paralysis, which resembles HyperKPP yet is mechanistically distinct, since the paralytic attacks are associated with a decreased plasma K+ levels [31]. The missense mutations causing HyperKPP affect the NaV1.4 channel activation and slow inactivation kinetics, while the fast inactivation kinetics remains unchanged [3336]. Normally, NaV1.4 becomes activated when EM reaches -50 mV with half-maximum activation at -15 mV and complete activation by 10 mV. Studies on the two most common mutations, T704M and M1592V in humans and the equivalent T695M mutation in rats, demonstrated that the steady state activation curve is shifted to more negative potentials with half-maximum activation at -30 mV and full activation at -10 mV [34]. One major consequence of such a shift is higher probability of generating action potentials when the cell membrane depolarizes. Like fast inactivation, slow inactivation prevents the opening of Na+ channels upon depolarization. The difference between the two processes is that fast inactivation occurs in ms while slow inactivation occurs over several seconds. Normally, none of the NaV1.4 channels are slow inactivated

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when EM is less negative than -100 mV. At the normal resting EM, between 15 and 25% of NaV1.4 are slow inactivated and full inactivation is observed at about -20 mV [35,33]. In HyperKPP muscles the steady state slow inactivation curve is shifted toward more depolarized EM and never reaches full inactivation as 10-20% of the defective NaV1.4 channels do not enter slow inactivation even at 20 mV. Furthermore, the mutations significantly slow down the rate of onset to and accelerate the recovery from slow inactivation. The major consequence of these changes is that the window of the steady state activation and slow inactivation curve is much larger; i.e., there is a greater range of EM for which the defective NaV1.4 channels can be activated while the slow inactivation does not occur, allowing greater open probability when the membrane is depolarized. A third defect has been found from analysis of single channel activity using myotubes from HyperKPP patients [37]. When the membrane is depolarized from -70 to -30 mV under voltage clamp, NaV1.4 channels briefly open for 1-2 ms and are fully fast inactivated within 4 ms. Raising [K+]e from 3.5 to 10 mM does not alter the inactivation kinetics. At 3.5 mM K+, most HyperKPP channels also inactivate within 4 ms whereas at 10 mM K+ channels changed to a dramatically altered gating mode. That is, some channels switch to a noninactivating mode giving rise to persistent opening throughout the depolarization step. While the mechanism is unknown, it appears to involve a direct effect of K+ on the defective NaV1.4 channels. It may be argued here that 10 mM is above the plasma [K +] observed during paralytic attacks; i.e., 6-8 mM. However, during exercise interstitial [K+] ([K+]int) is 4-5 mM above that of the plasma levels. Considering that the increase in [K+]int during paralysis is due to numerous action potentials as in exercise, it is then very likely that [K+]int reaches 10-13 mM during paralytic attacks. Taking into consideration all three major defects of NaV1.4 channels, i.e., steady state activation, slow inactivation and K+-induced inactivation mode, a mechanism can be proposed about how myotonic discharges and paralytic attacks are triggered. It has long being recognized that in normal muscles, there is a small Na+ permeability, being about 1% of the K+ permeability [38]. This is supported by the fact that addition of tetrodotoxin (TTX, a Na+ channel blocker) causes small hyperpolarization of the cell membrane and reduction in Na+ influx [1,39]. The TTX-sensitive Na+ influx (i.e., through NaV1.4 channels) is 6-times greater and resting EM is more depolarized in HyperKPP (-60 mv) than in normal (-80 mV) muscles [39]. Furthermore, the addition of TTX returns both Na+ influx and resting EM of HyperKPP muscles to values similar to those observed in normal muscles, confirming that the higher Na+ influx and lower resting EM in HyperKPP are actually due to defective NaV1.4 channels that have a higher opening probability than normal. These large membrane depolarizations associated with a steady state activation curve toward more negative potentials then facilitate the generation of action potentials leading to myotonic discharges [40]. As action potentials are generated, Kv channels are then activated allowing large K+ efflux to repolarize the cell membrane. As its extracellular concentration increases, K+ then causes some defective NaV1.4 channels to enter the

Renaud and Hayward

non-inactivating mode allowing even greater Na+ influx and greater depolarization, the latter also failing to increase the number of defective channels to enter slow inactivation. In normal muscles, an increase in [K+]e from 4 to 10 mM depolarizes the cell membrane from -80 to -65 mV with a rapid repolarization upon a return of [K+]e to 4 mM [41,1]. In HyperKPP muscles, the depolarization is much greater reaching -50 mV with a complete failure of repolarization when [K+]e is returned to 4 mM until TTX is added. These results again suggest that the excess depolarization is due to abnormal NaV1.4 channels at high [K+]e. More importantly, HyperKPP patients are heterozygotes as they have one normal and one abnormal SCN4A allele. The depolarization at 10 mM K+ observed in HyperKPP muscles is actually large enough to fast and slow inactivate all normal NaV1.4 channels and perhaps a fraction of the defective NaV1.4 channels [34,33]. The remaining available defective NaV1.4 channels are then too few to generate normal action potential resulting in paralysis. A final factor concerns the changes in intracellular [Na+] ([Na+]i) in HyperKPP muscles. Increases in [K+]e from 4 to 10 mM is not only associated with large membrane depolarization but also with almost a doubling of [Na+]i from 6 to 11 mM [41], a change large enough to reduce the action potential peak from 30 to 5 mV [42]. By itself such a change in [Na+]i is expected to cause less than 15% decrease in tetanic force [42]. However, when [Na+]i and [K+]e increases concomitantly, the depressing effects of each ion on force is not additive, but synergistic [43]. For example, while separate 1.8-fold decrease in Na+ concentration gradient and increase in [K+]e from 4 to 9 mM causes less than 10% decrease in M-wave amplitude (an index of membrane excitability) and tetanic force, the concomitant changes results in over 60% decrease in both M-wave (a measurement of membrane excitability) and tetanic force, which is well above the expected 20% if the effects were additive [44]. Thus, the paralysis can easily be a function of both the large depolarization and large increase in [Na+]i at high [K+]e. REGULATION OF MEMBRANE EXCITABILITY BY THE NA + K+ ATPASE PUMP, KATP AND CLC-1 CHANNELS In skeletal muscle, resting EM is a function of the concentration gradient of all ions that are permeable to the cell membrane and the activity of four major membrane components, the Na+ K+ ATPase pump, Kir2.1 inward rectifier, KATP and ClC-1 Cl- channels [2-4,6,45-47]. Recent studies have also demonstrated that the effects of a change in [K+]e on membrane excitability can be modulated by changes in the activity of the ClC-1 channels. So in this section, the mechanisms by which the pump, KATP and ClC-1 channels are controlled and affect membrane excitability will be discussed. In the next section, their potential as therapeutic targets to treat HyperKPP patients will be discussed. The Na+ K+ ATPase Pump It is well established that [K+]i decreases while [Na+]i and [K+]e increases during muscle activity and the changes become quite significant during fatigue development [19,4852]. As a consequence of these changes, the Na+ and K+ con-

Muscle Fatigue and Treatment of HyperKPP

centration gradient decreases. Lower Na+ concentration gradient reduces the Na+ diffusion rate during depolarization thereby reducing action potential amplitude [42]. A decrease in K+ concentration gradient, on the other hand, reduces the K+ equilibrium potential (EK) resulting in lower resting EM. As the resting EM depolarizes, the inactivation degree of NaV1.4 channels increases causing a reduction in the number of available channels, which also lowers action potential amplitude. As a consequence of the decrease in action potential amplitude, less Ca2+ is released and less force is generated [1,53]. The Na+ K+ ATPase pump is an important cell membrane component that opposes the decreases in Na+ and K+ concentration gradient by pumping three Na+ out and two K+ in for each ATP hydrolyzed. Furthermore, the unequal number of positive charges being pumped in and out results in the net efflux of one positive charge contributing to the hyperpolarization of the cell membrane. Several studies have clearly demonstrated the pump capacity in improving membrane excitability and force generation under conditions of elevated [K+]e or [Na+]i [54-59]. For example, force depression at moderate elevation of [K+]e can be fully reverse when the pump is activated with adrenergic agonists or slight increase in stimulation rate. The pump can also reduce the rate at which force decreases when [K+]e is increased. More importantly, there is at least one study demonstrating an activation of the pump during muscle activity in the absence of any increase in [Na+]i [60]. In other words, muscle fibers have the capacity to prevent Na+- and K+-induced force depression by activating the pump. The KATP Channel The KATP channel is a voltage-independent, weak inward rectifier and ligand dependent K+ channel. Its name derived from the fact that the channel is closed by the binding of ATP [61]. ATP is, however, not the only ligand that modulates the channel activity. In fact, the channel is activated by the same changes in metabolites that occur during metabolic stresses such as fatigue or ischemia. That is, it is activated by decreases in intracellular ATP and pH, increases in intracellular ADP and extracellular adenosine [61-64]. Thus, the channel behaves as an energy sensor becoming active during a metabolic stress causing a decrease in energy availability. One major function of the channel is to prevent fiber damage during exercise in both cardiac and skeletal muscles [65-69]. Two mechanisms have so far been elucidated by which the channel prevents fiber damage. The first mechanism involves a direct effect on action potential. Being voltage independent, once activated the KATP channel remains opened at all EM. Consequently, it allows for greater K+ efflux that counteracts the Na+-induced depolarization. In unfatigued skeletal muscle, an activation of KATP channels with pinacidil causes a decrease in overshoot by 15 mV [70,71] and this effect is accentuated during fatigue development when more KATP channels are activated [70]. As action potential amplitude decreases less Ca2+ is released and less force is generated. It has been hypothesized that this effect is crucial to reduce the activity of Ca2+ ATPase pump and myosin ATPase in order to preserve ATP

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since large depletion can cause fiber damage and even cell death. The second mechanism involves the maintenance of resting EM. Normally, during fatigue resting EM depolarizes by 10-15 mV in both amphibian and mammalian muscles, while during metabolic inhibition it does not change in amphibian muscles [72-75]. When KATP channels are blocked with glibenclamide, the depolarization becomes excessive in many fibers; it can be as large as 50 mV, bringing resting EM close to -30 mV [72,73]. There is now clear evidence that this depolarization is large enough to activate CaV1.1 channels allowing excessive Ca2+ influx and increases in myoplasmic [Ca2+] ([Ca2+]i) even when muscles are not stimulated to contract [72,76]. This increase in [Ca2+]i has two major consequences; i) it further increases the metabolic stress lowering energy levels because higher [Ca2+]i increases the activity of the Ca2+ ATPase pump and myosin ATPase activity, and ii) chronic increase in [Ca2+]i is known to result in fiber damage [77,78]. Overall, KATP channels have two major effects on membrane excitability. They lower action potential amplitude by providing a K+ efflux to reduce the depolarization. They are also very effective in preventing cell membrane depolarization as large as 50 mV in the resting state. Modulation of the K+-Induced Force Depression by the ClC-1 Cl- Channels It is well established that [K+]e increases during exercise and because of its depressive effect on membrane excitability and force development the increase has long been considered a major factor in the etiology of muscle fatigue. However, even a muscular activity that does not lead to fatigue, such as one leg bicycling exercise at 30 watt for 30 min, causes an increase in [K+]int that can reach 10-12 mM within 5 min. In vitro, such increases in [K+]e causes major decreases in force development, raising the issue as to how one can bicycle for 30 min. The answer involves the Na+ K+ ATPase pump as discussed above and ClC-1 Cl- channel, which will now be discussed. One major function of the channel is to clamp resting EM. For example, the depolarization associated with an increase in [K+]e is much greater and faster in the absence of Cl- (to abolish Cl- conductance, GCl) than in the presence of Cl- in the physiological solution bathing muscle fibers [45]. From this point of view, one would expect that any decrease in GCl worsens the K+-induced force depression because of greater membrane depolarization. However, while the ClC-1 channel is voltage sensitive, its kinetics of activation and deactivation are too slow to result in any changes in GCl during an action potential [79]. Consequently, there is a constant Cl- influx during action potential that counteracts the Na+-depolarization phase while helping with the K+-induced repolarization phase. At normal [K+]e, removal of all Cl- in the physiological solution has little effect on action potential depolarization phase [80]. The lack of any effect is most likely because of the relatively much larger Na+ conductance (GNa) that easily overcomes the Cl- effect. The situation is, however, quite different at elevated [K+]e. This is because under those con-

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ditions the degree of NaV1.4 inactivation is elevated resulting in a much lower GNa for action potential generation. For example, at 11 mM K+, about 50% of muscle fibers are unexcitable and those that remained excitable have on average an EM of -10 mV at the peak of the action potential [81]. When a small but incomplete decrease in GCl is induced by lowering extracellular [Cl-] or exposing fibers to 9-AC, a ClC-1 channel blocker, the number of excitable fibers increases to 90% while the action potential peak increases to 10 mV. Associated with an increase in membrane excitability is an increase in force development. In fact, small decrease in GCl shift the [K+]e-force relationship toward higher [K+]e; i.e., it reduces the K+-sensitivity of muscle [81]. The effects of changing GCl on the K+-induced membrane excitability and contractility discussed above were studied in unfatigued muscles. Two recent studies by Pedersen et al. [6,7] have now reported small increases in GK and about 70% decreases in GCl at the onset of action potential firing, the latter change involving the phosphorylation of ClC-1 channels by protein kinase C. These two studies are quite significant because they demonstrate that the modulation of the K+-induced force depression by GCl observed in unfatigued muscle is physiologically relevant. More importantly, a decrease in GCl may be the reason as to why the biking exercise mentioned above could last 30 min despite large increases in [K+]int; i.e., the decrease in GCl may have prevented any decrease in membrane excitability and contractility when [K+]int reached levels exceeding 10 mM. Finally, the same two studies also showed that after continuous action potential firing, the activity of both ClC-1 and KATP channels suddenly increases far above that observed in unfatigued muscle fibers. The time of such an increase appears to depend on the intensity of the stimulation and on glucose. That is, the higher the intensity or the removal of extracellular glucose reduces the time interval between the start of the stimulation and the time the activity of both channels increases. Considering that both channels are ATP dependent [61,82] and that the KATP channel is known to be activated during metabolic stress, it is then very likely that those channels are activated to reduce membrane excitability; i.e., higher GCl will increase muscle sensitivity to the K+-induced force depression while higher KATP channel activity will further reduce action potential amplitude; the ultimate goal being the reduction of ATP usage by the Ca2+ ATPase and myosin ATPase. Overall, these studies clearly demonstrated that the activity of the Na+ K+ ATPase pump, ClC-1 and KATP channels are regulated during muscle activity. The changes at the onset of exercise allow for a maximization of muscle performance such as the increase in Na+ K+ ATPase pump and decrease in GCl to prevent any K+-induced depression of membrane excitability. When a metabolic stress occurs, changes in the activity of the same ion channels occur with the aim of reducing membrane excitability and contractility to preserve ATP. So, the final question concerns how we can use the Na+ K+ ATPase pump, KATP and ClC-1 Cl- channels to develop new and more effective therapeutic approaches to alleviate HyperKPP symptoms. The answer may come from the mechanisms that increase membrane excitability.

Renaud and Hayward

THE NA + K+ ATPASE PUMP, KATP AND CLC-1 CHANNELS AS POTENTIAL TARGET FOR NEW AND MORE EFFECTIVE PHARMACOLOGICAL STRATEGIES TO TREAT HYPERKPP PATIENTS When HyperKPP patients ingest KCl or rest after exercise plasma [K+] increases to 7-8 mM while the force generated by muscle decreases by over 80% [17,83]. Both the large increase in plasma K+ and the decrease in force are prevented when patients are treated with the adrenergic agonist salbutamol to activate the Na+ K + ATPase pump. Recently, a HyperKPP mouse model was generated in which the equivalent human M1592V mutation was ‘knocked in’ the mouse genome. Muscles from this HyperKPP mouse have several of the symptoms observed in human, including large TTX-dependent Na+ influx, depolarized cell membrane and large decrease in force when [K+]e is elevated [39,84]. Treating soleus from HyperKPP mice with salbutamol allowed for full recovery of force at 10 mM K+ to level similar to those in wild type soleus [39]. An activation of Na+ K+ ATPase pump by salbutamol improves force generation in HyperKPP muscles most likely because it i) helps repolarizing the cell membrane and ii) lowers [Na+]i. However, by itself the salbutamol treatment is not sufficient as it loses its effectiveness over time in some but not all HyperKPP patients [16]. The KATP channel is another membrane component to consider. The idea here is to take advantage of the capacity of the channel to prevent membrane depolarization as large as 50 mV. As discussed above, the myotonic discharges are due to defective NaV1.4 that allows large Na+ influx. An activation of KATP channel (or any increases in GK) may help in counteracting the Na+-induced membrane depolarization eliminating or at least decreasing the frequency of myotonic discharges. Fewer action potentials produce smaller increases in [K+]e despite the increase in GK. This is because while K+ efflux is high during action potential, it is very low in the resting state since EK is close to resting EM. The ClC1 channel can also be a target where in this case we are taking advantage that small decreases in GCl reduce the K+sensitivity for force depression. So, if an activation of KATP channel activity is insufficient to fully prevent myotonic discharges, a concomitant treatment that lowers GCl may help prevent the paralytic attacks that are caused by elevated [K+]int. Of course, such pharmacological approaches must take into consideration the adverse effect of activating KATP channels and decreasing ClC-1 channel activity. That is, the direct depression of action potential amplitude by the former and the greater membrane depolarization at elevated [K+]e of the latter. Here, it is hypothesized that small increase in GK may help in preventing large membrane potential while only large increase in activity would be detrimental due to the direct effect on action potential. This is supported by the fact that an activation of KATP channels in unfatigued muscles has no effect on force generation despite a 15 mV (from 30 to 15 mV) decrease in action potential overshoot [1]. It is also because peak twitch force is not affected unless the action potential overshoot becomes less than 5 mV [1,42]. In fact, a large decrease in membrane excitability and perhaps force occurs when abundant KATP channels are activated

Muscle Fatigue and Treatment of HyperKPP

such as during fatigue [70]. In other words, there seems to be a margin of safety in which a small decrease in action potential amplitude does not affect force development. Similarly, at elevated [K+]e a small decrease in GCl improves action potential generation while a very large decrease has the opposite effect because the larger K+-induced membrane depolarization overrides the improvement of action potential with lower GCl. In support of this possibility is the fact that a small decrease in GCl improves force development at elevated [K+]e [81] and only complete removal of GCl increases the rate of force decline during fatigue as [K+]e increases [85]. Finally, one cannot ignore the possibility that the increase in action potential amplitude with lower GCl will also help counteracting the decrease associated with an activation of KATP channels.

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However, as discussed above, such treatment only alleviates HyperKPP symptoms and eventually loses effectiveness over time. Therapy for Hyperexcitability Diseases (Patent US20100324144, Heers et al., December 23, 2010) [89]

Treatment involving a modulation of ClC-1 and KATP channels may still, however, be insufficient because while it helps with improving membrane excitability, it does nothing in regard to the decrease in action potential amplitude related to large increases in [Na+]i. Concomitant activation of the Na+ K+ ATPase pump may be required to help not only in reducing [Na+]i but also in preventing membrane depolarization.

This patent is for the discovery of a class of peptide compounds to treat disease that causes membrane hyperexcitability and for class of diseases associated with defective ion channels. Some of the peptides have been used to treat diseases like epilepsy and chronic pain, which are diseases of the nervous system. The compound lacosamide enhances slow inactivation by shifting the steady state inactivation curve toward more negative membrane potential of normal NaV1.4 channels while having no effect on the activation and fast inactivation kinetics. Lacosamide has so far not been tested as a treatment for any hyperexcitability disorders. While it has the potential in helping patients suffering of HyperKPP, it remains to be determined if it will be fully effective because while it can help restoring a normal steady state slow inactivation it may reestablish a normal steady state activation or prevent high concentration of K+ to trigger the non-inactivating mode of the channel.

PATENTS

CURRENT AND FUTURE DEVELOPMENTS

Methods of Detecting Periodic Paralysis in Horses (Patent US5356777, Hoffman et al., October 18, 1994) [86]

Overall, HyperKPP involves two opposing events at the level of the muscle cell membrane; one being the myotonic discharges or the generation of uncontrolled action potentials; the other one being the paralysis which is a complete loss of membrane excitability. A review of patents also revealed one potential new treatment for HyperKPP patients, but the drug, Lacosamide, remained to be tested. Here, we also propose to test in future studies a new therapeutic approach to treat HyperKPP patients. This approach involves the concomitant activation of Na+ K+ ATPase pumps and KATP channels with a partial inhibition of ClC-1 channels. The pump and KATP channel stimulation is expected to counteract the Na+-induced membrane depolarization by hyperpolarizing the cell membrane and thus the frequency of myotonic discharges. Furthermore, the pump would also counteract the increase in [Na+]i, and [K+]e that is associated with action potential. Finally, a reduced activity of the ClC-1 channels may act synergistically to generate stronger action potentials at elevated [K+]e preventing any paralysis.

This patent relates to the discovery of a mutation causing HyperKPP in horses and for the development of a diagnostic test. The invention comprises two oligonucleotide probes, one for normal and one for HyperKPP horses, which are used for the polymerase chain reaction (PCR) reaction. In this case, the mutation is a phenylalanine to a leucine mutation in domain IV transmembrane segment S3 of the NaV1.4 channel. Methods and Systems for Universal Carrier Screening (Patent US20100022406, Srinivasan et al., January 28, 2010) [87] For this more recent patent, a method was developed for genetic screening. The idea is to use the genetic screening of two or more individuals to predict the phenotype of a child from the same group of individuals. The method is not restricted to just HyperKPP as it can be used for several other diseases, especially for rare diseases like HyperKPP. Furthermore, the method works for more than one mutation including the two most common one, T704M and M1592V. The test relies heavily on probability following the genetic screening of some individuals. Methods for Treating Carbonic Anhydrase Mediated Disorders (Patent US20030220376, Masferrer et al., November 27, 2003) [88] This patent discusses the use of several compounds that inhibit the activity of carbonic anhydrase which catalyzes the reaction between carbon dioxide and water to produce bicarbonic acid. There are several diseases which can be treated by inhibiting carbonic anhydrase including HyperKPP.

CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS Declared none. REFERENCES [1] [2]

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