George Neurobio of Disease

Brittany Adler 3/14/07

Channelopathy Diseases
A diverse array of channelopathies, which are defects in ion channels caused by genetic mutations in the ion channel genes, have been identified to date. Over 40 human diseases are attributable to channelopathies. The numerous ion channels that can be affected by genetic mutations allow for this diversity in disease phenotypes. Channelopathies have been identified in the Na+, K+, Cl- and Ca2+ voltage-gated ion channels, as well as in the nACh, Gly, and GABA ligand-gated channels. Moreover, different mutations with distinct functional defects can occur within a single membrane channel, thereby contributing to the diversity of phenotypes expressed. All channelopathies alter the membrane excitability of cells, whether in the skeletal muscle or in the brain. This often results in phasic symptoms in which a brief episode of dysfunction is followed by normal activity. Typically a channelopathy disease affects only one organ because membrane channel expression is tissue-specific. The periodic paralyses disorders are characterized by muscle weakness and abnormal potassium concentrations in serum. Patients afflicted with hyperkalemic periodic paralysis (HyperPP) have elevated levels of blood potassium, whereas those with hypokalemic periodic paralysis (HypoPP) have suppressed potassium levels. Difficulty in relaxing the muscles after a voluntary movement (myotonia) is also commonly observed in patients with HyperPP but not HypoPP. Episodes of muscle weakness in HyperPP patients are often triggered by cold or events that raise blood potassium, such as rest after exercise, fasting, or a high potassium diet. Although it is unclear how potassium initiates the attacks of muscle weakness, evidence suggests that defects in the muscle Na+ channel are primarily responsible for the disorder. The marked membrane depolarizations observed in HyperPP patients after an increase in extracellular potassium could not

be solely attributable to a change in potassium conductance. Moreover, a small increase in extracellular potassium induces an abnormally large Na+ current in HyperPP patients that is reversed by TTX administration. Fontaine et al. explored the role of the Na+ channel α subunit in HyperPP by testing for a genetic linkage between the HyperPP mutation and the DNA region that codes for the α subunit. To do this, the researchers first mapped the location of the α subunit in normal people. Regions on human DNA that contain the adult human α subunit and the fetal human α subunit were identified using the probes Na2 and Na3, which are sequences of base pairs that are highly homologous to the adult and fetal human α subunits, respectively. Na2 and Na3, which both contain the adult human α subunit region (Na3 contains the fetal α subunit as well) were identified on chromosome 17. Interestingly, when these probes were tested on DNA from 5 human subjects, a restriction fragment length polymorphism Bgl II RFLP was found on chromosome 17. The researchers estimated that there was less than a 1 in 107 chance that Na2 was located near the growth hormone (GH1) region by chance alone. This suggests that GH1 is tightly linked to Na2 and can also be used as a genetic marker of the α subunit. When GH1 and Na2 were used as markers in DNA obtained from blood samples from a family afflicted with hyperPP, the results agreed with the pedigree and showed that the three regions GH1, Na2 and HyperPP were tightly linked in HyperPP family members. Because the α subunit region is genetically linked with the HyperPP mutation, it is likely that the primary dysfunction in HyperPP patients is in the α subunit of the Na+ channel. Cannon et al. explored the mechanism by which a sodium channel defect can induce the symptoms of HyperPP after an increase in extracellular potassium. As mentioned above, previous studies suggested that the dysfunction may be caused by depolarization of the cell membrane, particularly by an increase in Na+ conductance. To explore this hypothesis further, Cannon et al.

compared single-channel recordings from myotubes isolated from a person with HyperPP and from a normal person. The single-channel conductances measured after a depolarizing current were the same for the excised HyperPP and wild type channels. However, the single-channel currents in the two types of muscle were different. Although there was no difference in the mean open time of Na+ channels in 3.5 mM K+ after a depolarizing step, some of the HyperPP channels stayed open significantly longer than normal channels in 10 mM K+ after a depolarizing current. These results suggest that the HyperPP channels lose their ability to inactivate during a sustained depolarization. Similar results were obtained when single-channel currents were recorded from patches still attached to the cell, even for a mild depolarizing step to -40 mV. Based on observations of only a few noninactivating channels opening late, the researchers determined that there is a low probability that a channel will not be able to inactivate. The researchers measured the probability of noninactivation and found that it increased with extracellular K+ concentration in HyperPP cells but not in normal cells. K+ probably acts on the Na+ channel directly to trigger it to become noninactivating. Because Na+ channels in HyperPP myotubes can become noninactivating if they are exposed to a solution that blocks K+ conductance and inhibits second messengers, it is unlikely that the effects of K+ are mediated by a second messenger system. Interestingly, a positive feedback system exacerbates the effects of K+ on Na+ channels. The K+-induced opening of noninactivating Na+ channels depolarizes the cell membrane, thereby causing K+ to flow out of the cell, which increases the extracellular K+ concentration further. At a certain point the membrane potential becomes sufficiently depolarized that normal Na+ channels become inactivated. This makes the cell less responsive to subsequent stimuli and results in the muscular dysfunction commonly observed in HyperPP patients. Furthermore, if fewer channels are noninactivating, the membrane potential may be slightly depolarized so that it is closer to threshold

but does not inactivate normal channels. This situation may increase firing of the neuron and cause the symptoms of myotnia in some patients. It is unclear how the mechanisms underlying HyperPP and HypoPP induce the same symptoms of muscular weakness. Previous studies have found that HypoPP is associated with mutations in the α1 subunit of the dihydropyridine (DHP) receptor, which controls the L-type Ca2+ current and acts as a voltage-gated Ca2+ channel. HypoPP patients have mutations in the gene CACLN1A3, which may disrupt the voltage-gating function of DHP by reducing the positive charge of arginine. Histidine is substituted for arginine in segment IIS4 in some patients (R528H), while others have a substitution of histidine or glycine for arginine in segment IVS4 (R1239H). Sipos et al. tested 24 HypoPP families and found mutations in myotube mRNA in 9 of the families. Using whole-cell recording and a patch clamp-amplifier, the researchers examined how the current– voltage relationship and inactivation pattern differed for slow L-type currents, T-type currents, and fast Ca2+ inward currents (“third type currents”) in myotubes of patients expressing the mutations relative to controls. The L-type current in myotubes isolated from patients with the R528H mutation had a normal current-voltage relationship but an abnormal inactivation curve. Inactivation of the L-type current occurred at more negative potentials relative to the control. Furthermore, there was a 26% increase in cells expressing the third type current in R528H mutant myotubes, which lead to a stronger current at more positive membrane potentials. In contrast, the R1239H mutation did not affect the inactivation curve of the L-type current, but it did reduce the strength of this current at membrane potentials from 0 to +50 mV. The current density was not voltage-dependent, so it was not reduced in R1239H cells by channel inactivation during the holding potential at -90 mV. Although the inactivation curve for the third type current in R1239H mutant myotubes did not differ from controls, 41% more cells expressed this current, resulting in a similar voltage-current curve as

that determined for the R528H mutants. These results suggest that the mutations implicated in HypoPP cause abnormal Ca2+ currents. It is unclear if the third type current is mediated by mutant L-type channels. The altered activity of L-type currents in the mutant myotubes is likely caused by the mutations in the α1 subunit of DHP receptors. Interestingly, the abnormal inactivation curve of the L-type current suggests that the effects may be caused by increased inactivation of the slow L-type channel. It is possible that these mutations reduce Ca2+ release, thereby causing muscle weakness. A mutation in the Na+ channel gene SCN4A that replaces a histidine with an arginine in domain IIS4 (R669H) has also been identified in several HypoPP patients. Struyk et al. introduced this mutation into a human kidney cell line and studied the voltage gating properties of the mutant Na+ channels using whole-cell recording. The researchers measured fast-inactivation by finding the time constant τ, or the rate of recovery of the Na+ current from fast inactivation after a conditioning pulse. Fast inactivation was not different in the mutant relative to the wild type channels. This is consistent with previous studies that have shown that domains I and II of the Na+ channel are not affected by fast inactivation. However, slow inactivation was significantly greater in the R669H channels than in wild types. A conditioning pulse was applied to cells held at -120 mV, followed by a restoration period at the holding potential to allow fast inactivation to recover. During a test pulse at -10 mV, the Na+ current was smaller in R669H channels after long depolarized conditioning periods, which suggests that the mutant channels have enhanced slow inactivation. After a 40 sec conditioning pulse, when slow inactivation had reached steady state, the mutant channels had a hyperpolarized shift in the voltage-current curve of 10 mV, which is indicative of increased slow inactivation. When channel recovery from the inactivating state to the resting state was tested after a 40 msec, 1 sec, and 60 sec conditioning pulse, the researchers found that few channels were slow-

inactivated at 40 msec, and there was little difference between the R669H and wild type channels. However, intermediate-inactivated channels (IM), which have a kinetic profile in-between that of slow- and fast-inactivated channels, had a significantly slower recovery after a 1 sec pulse in mutant channels. Slower recovery of the mutant IM channels and IS channels (slow-inactivating channels) was also observed after a 60 sec and 240 sec conditioning pulse. Overall, R669H channels had a recovery period that was five times longer for IM channels and two times longer for IS channels relative to the wild type. These prolonged recovery periods from inactivation support the hypothesis that mutant Na+ channels have enhanced slow inactivation. The gating defects in R669H channels were not affected by changing the extraellular K+ concentration. Although the results clearly suggest that mutant Na+ channels have enhanced slow inactivation, it is unclear how this defect causes the symptoms of HypoPP. The finding that a deficit in Na+ channel inactivation causes similar symptoms of muscle weakness in HyperPP complicates the picture further. Considering that HypoPP cells are depolarized when extracellular potassium is reduced, it is likely that a membrane depolarization is responsible for reducing muscle excitability. More studies need to be done to understand the underlying mechanisms of HpyerPP and HypoPP so that their similar behavioral effects can be reconciled.