Expression patterns of two potassium channel genes in skeletal muscle cells of patients with familial hypokalemic periodic paralysis
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.84331
Source of Support: None, Conflict of Interest: None
Background: Familial hypokalemic periodic paralysis is an autosomal-dominant disorder characterized by episodic attacks of muscle weakness with hypokalemia. The combination of sarcolemmal depolarization and hypokalemia has been attributed to abnormalities of the potassium conductance governing the membrane potential; however, the molecular mechanism that causes hypokalemia has not yet been determined. Aim: To test the hypothesis that the expression patterns of delayed rectifier potassium channel genes in the skeletal muscle cells of patients with familial hypokalemic periodic paralysis differ from those in normal cells. Material and Methods: We examined both mRNA and protein levels of two major delayed rectifier potassium channel genes KCNQ3 and KCNQ5 in the skeletal muscle cells from three patients with familial hypokalemic periodic paralysis and three healthy controls. Results: When normal cells were exposed to 50 mM potassium buffer, which was used to induce depolarization, the KCNQ3 protein level significantly increased in the membrane fraction but decreased in the cytosolic fraction, whereas the opposite was true in patient cells. Conclusion: Abnormal subcellular distribution of the KCNQ3 protein was observed in patient cells. Our results suggest that the altered expression of KCNQ3 in patient cells exposed to high extracellular potassium levels could possibly hinder normal function of the channel protein. These findings may provide an important clue to understanding the molecular mechanism of familial hypokalemic periodic paralysis.
Keywords: Genetic diseases, neuromuscular diseases, paralysis
Familial hypokalemic periodic paralysis (HOKPP) is an autosomal-dominant channelopathy characterized by reversible attacks of flaccid paralysis of the skeletal muscles with concomitant hypokalemia. Onset of the disease is in the second decade of life, and usually presents during adolescence. Paroxysmal muscle weakness mainly develops at night or early in the morning and is frequently triggered by emotional stress, meals rich in carbohydrates and salt, strenuous exercise, or exposure to cold. , Recent molecular studies have revealed that the majority of HOKPP cases are caused by mutations in the human skeletal muscle voltage-gated calcium channel gene CACNA1S or sodium channel gene SCN4A; however, the mechanism that causes hypokalemia has not been clearly elucidated. ,,
The KCNQ family of voltage-gated potassium channels mediates the delayed rectifying potassium current that plays a critical role in the regulation of excitability of electrically active cells such as myocytes and neurons. As of now, the KCNQ family consists of five members, all of which are differentially expressed in specific tissues/organs and associated with human diseases. ,,, KCNQ5 shows widespread expression in skeletal muscle, where the channel generates potassium currents that contribute to repolarization of cell, terminating the action potential. The potassium channel protein encoded by KCNQ5 has been demonstrated to interact with the potassium channel subunit encoded by KCNQ3 to form functional heteromeric channels. ,
Previous studies have suggested a relationship between the development of HOKPP and decreased potassium channel function of skeletal muscle cells. , This study was conducted in order to test the hypothesis that the expression patterns of major delayed rectifier potassium channel genes KCNQ3 and KCNQ5 in skeletal muscle cells of HOKPP patients differ from those in normal cells. We chose to study their expression because of their key role in regulating the movement of potassium ions through cell membranes in skeletal muscle.
We reviewed 178 patients who were being treated for HOKPP in the Department of Pediatrics, Konyang University Hospital. For this study, we selected three patients who presented with the most severe symptoms. These patients had the Arg1239Gly mutation in CACNA1S. Three healthy individuals participated in the study as controls. All participants had given written informed consent, and the study was conducted in compliance with the guidelines of the Institutional Review Board of the Konyang University Hospital.
Sampling of skeletal muscle specimens
Subjects were asked to rest in a supine position on a bed. Skeletal muscle specimens were collected from the gastrocnemius muscles through a surgical incision following local anesthesia with lidocaine.
Preparation of potassium buffers
We prepared a 4 mM potassium buffer at pH 7.35 (4 mM KCl, 145 mM NaCl, 1 mM MgCl 2 , 0.5 mM CaCl 2 , 5 mM glucose, and 10 mM 3-(N-morpholino) propanesulfonic acid (MOPS)) to expose cells to normal extracellular potassium concentrations. To trigger depolarization of skeletal muscle cells under a high concentration of potassium, 50 mM potassium buffer (50 mM KCl, 145 mM NaCl, 1 mM MgCl 2 , 0.5 mM CaCl 2 , 5 mM glucose, and 10 mM MOPS) was prepared. Both solutions were sterilized prior to experimental use.
Cultivation of skeletal muscle cells and treatment with potassium buffer
Cultivation and differentiation of skeletal muscle cells were performed using a previously described protocol.  Briefly, after pretreatment, skeletal muscle specimens collected from HOKPP patients and healthy controls were cultured using Dulbecco's modified Eagle's medium (DMEM; Thermo Scientific, South Logan, UT, USA) containing 20% fetal bovine serum (Thermo Scientific) and 1% penicillin-streptomycin (Thermo Scientific) at 37°C in an incubator containing 95% air and 5% CO 2 (Thermo Scientific). Thereafter, skeletal muscle cells were cultured in DMEM with 2% horse serum (Thermo Scientific) and 1% penicillin-streptomycin and allowed to differentiate for 5 d. Both the normal and patient cells were collected at the tenth passage and used for analysis. mRNA and protein levels of KCNQ3 and KCNQ5 in skeletal muscle cells from both normal and patient groups were analyzed prior to and 1 h following addition of potassium buffers.
Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from cultured skeletal muscle cells using TriZol (Invitrogen, Carlsbad, CA, USA), of which 100 ng was converted to cDNA with reverse transcriptase. AccuPower PCR PreMix (BIONEER, Daejun, Korea) was added to the reaction mix, and quantitative RT-PCR analysis was performed using primers for KCNQ3: forward 5′-CTC AGC AAC AAC GTA TGT GG-3′, reverse 5′-GAA TCA GAA ATC CCA TCC CC-3′, and KCNQ5: forward 5′-GTC AAA TCT CAC CAA GGA CC-3′, reverse 5′-GGC ATC TGT ACT TTC TCC TG-3′. Quantitative measurement of mRNA was obtained from 10 independent experiments. The expression levels of mRNAs specific for each gene were normalized to the expression of GAPDH (glyceraldehyde 3-phosphate dehydrogenase).
Western blot analysis
Skeletal muscle cells obtained from patients and healthy controls were treated with two differing concentrations of potassium buffers for 1 h, and cytosolic and membranous protein fractions were then separated. A modification of a cell separation method described previously was used for the separation of cytosolic and membranous proteins , and a protease-inhibitor cocktail (Sigma, Saint Louis, MO, USA) was added at each step to extract the proteins. From each specimen, 20 μg protein was electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad, Hercules, CA, USA), and then transferred to a polyvinylidene fluoride membrane (Bio-Rad) for Western blot analysis. Protein-containing membrane was blocked using 5% skim milk (Bio-Rad) and then incubated with primary anti-KCNQ3 and anti-KCNQ5 antibodies (Abcam, Cambridge, MA, USA). Subsequently, anti-rabbit and anti-goat secondary antibodies were used, and the protein band was then visualized with SuperSignal West Pico Luminal/Enhancer Solution (Pierce, Rockford, IL, USA). Quantitative densitometric analysis of Western blot assays for KCNQ3 and KCNQ5 proteins was obtained from 10 independent experiments.
SPSS Version 19.0 Software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Data were presented as means ± standard deviation. Comparisons were made using two-way ANOVA followed by Mann-Whitney U test. A P value <0.05 was considered statistically significant.
mRNA expression of KCNQ3 and KCNQ5 potassium channel genes
We used quantitative RT-PCR to examine the mRNA levels of the two potassium channel genes KCNQ3 and KCNQ5 in skeletal muscle cells from both normal controls and patients prior and after exposure to 4 mM and 50 mM potassium buffers. No change in the mRNA level of KCNQ3 or KCNQ5 was observed in normal control or patient cells following exposure to 4 mM potassium buffer (data not shown). Similarly, neither normal control nor the patient cells demonstrated detectable changes in expression of the two genes following exposure to 50 mM potassium buffer (data not shown).
Western blot analysis to evaluate membranous and cytosolic expression of KCNQ3 and KCNQ5
We investigated protein expression level of KCNQ3 and KCNQ5 in both normal control and patient cells prior and after exposure to 4 mM and 50 mM potassium buffers. Membranous and cytosolic fractions of the cells were separated and protein expression levels of KCNQ3 and KCNQ5 were evaluated. When normal control and patient cells were exposed to 4 mM potassium buffer, no quantitative change in the protein level of KCNQ3 and KCNQ5 was observed in the cytosolic fraction of treated cells relative to that of untreated cells [Figure 1]a and [Figure 2]a. However, protein level of KCNQ3 in the cytosolic fraction decreased in normal control cells but increased in patient cells after exposure to 50 mM potassium buffer [Figure 1]b and [Figure 2]b. Interestingly, the protein level of KCNQ3 in the membrane fraction increased in normal control cells but decreased in patient cells after exposure to 50 mM potassium buffer [Figure 3]. The protein level of KCNQ5 did not vary in either cell type after exposure to 50 mM potassium buffer.
After intracellular sodium ions trigger an action potential, delayed rectifier potassium channels discharge intracellular potassium ions to the exterior, restoring the membrane potential to resting status. It has been assumed that hypokalemia of paralytic attacks in patients with HOKPP is the result of potassium ions pooling inside the cell because they cannot be freely discharged; however, the molecular mechanism has not yet been determined.  Previous studies have revealed a relationship between the development of HOKPP and decreased potassium channel function of skeletal muscle cells. , Unlike inward rectifier potassium channels that are primarily involved in stabilizing the resting membrane potential in skeletal muscle cells, delayed rectifier potassium channels are essential for repolarization following depolarization of the cell membrane; this process is presumed to be defective during paralytic attacks in HOKPP. Recently, through a molecular study of skeletal muscle cells from patients with this disorder, we suggested that hypokalemia may be attributable to potassium channel abnormalities.  In the present study, we examined this possibility further by investigating the variation in gene expression for major delayed rectifier potassium channels in skeletal muscle cells from normal and patient groups.
Quantitatively, no change in the mRNA expression for KCNQ3 and KCNQ5 was observed for either group following exposure to 4 mM and 50 mM potassium buffers; the former was used to mimic normal extracellular potassium concentrations and the latter to induce depolarization and to mimic paralytic conditions. In addition, when normal control and patient cells were exposed to 4 mM potassium buffer, no quantitative change in the protein level of KCNQ3 and KCNQ5 was observed in treated or untreated cells. This is consistent with patients experiencing no difficulty in carrying out ordinary activities in between the attacks. However, when normal cells were exposed to 50 mM potassium buffer, the KCNQ3 protein level decreased in the cytosol and increased in the cellular membrane. Given that delayed rectifier potassium channels are activated by the influx of sodium ions and counteract their effect by allowing the discharge of potassium ions in normal skeletal muscle cells, this is presumably a mechanism to stabilize cells to a resting membrane potential by resolving the depolarization. In contrast, KCNQ3 protein expression increased in the cytosol but decreased in the membrane when the patient cells were exposed to 50 mM potassium buffer. These results imply that reduced expression of the KCNQ3 potassium channel subunit in the depolarized membrane of patient cells is so dysfunctional that intracellular potassium ions are not discharged, but pool inside the membrane, resulting in a prolonged state of depolarization and ultimately leading to clinical hypokalemia and paralysis. Abnormal trafficking/localization of channel proteins has been implicated in the pathogenesis of many human diseases. ,, Future work on other channel proteins that are functionally related to KCNQ3 will further explore this possible mechanism of hypokalemia and may reveal how a calcium or sodium channel gene mutation can lead to altered expression of other genes.
In conclusion, this study examined the expression patterns of two major delayed rectifier potassium channel genes in the skeletal muscle cells of HOKPP patients. We observed abnormal subcellular distribution of the KCNQ3 protein in patient cells. This is a novel finding that explains the pathogenesis of this disease with regard to delayed rectifier potassium channels in patient cells.
[Figure 1], [Figure 2], [Figure 3]