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Clin Transl Sci. Author manuscript; available in PMC 2010 April 14.
Published in final edited form as:
PMCID: PMC2854805

A Patient Suffering from Hypokalemic Periodic Paralysis Is Deficient in Skeletal Muscle ATP-sensitive K+ channels


Hypokalemic periodic paralysis (HOPP) is a rare disease associated with attacks of muscle weakness and hypokalemia. In the present study, immunoprecipitation/Western blotting has shown that a HOPP patient was deficient in sarcolemmal KATP channels. Real-time RT-PCR has revealed that HOPP has decreased mRNA levels of Kir6.2, a pore-forming KATP channel subunit, without affecting the expression of other KATP channel-forming proteins. Based on these findings, we conclude that HOPP could be associated with impaired expression of Kir6.2 which leads to deficiency in skeletal muscle KATP channels, which may explain the symptoms and clinical signs of this disease.

Keywords: hypokalemic periodic paralysis, KATP channels, skeletal muscle, Kir6.2


Hypokalemic periodic paralysis (HOPP) is a debilitating disease characterized by reversible attacks of muscle weakness accompanied with episodes of hypokalemia. It is well established that the majority of familial HOPP is due to mutations in a skeletal muscle voltage-dependent Ca2+ channel, mainly dihidropyridine receptor. However, it is intriguing that the main symptoms/signs of this disease, including hypokalemia, depolarization of muscle fibers, and paralysis following insulin administration, cannot be explained by the mutation of Ca2+ channels.1 In animal experimental models, hypokalemia and paralysis are usually seen when permeability of sarcolemma to K+ is impaired.2,3 Electrophysiological measurements have shown that HOPP patients have decreased K+ current flowing through sarcolemmal ATP-sensitive K+ (KATP) channels in skeletal muscles.4

KATP channels are gated by intracellular ATP, and this ion channel couples metabolic state of the cell with membrane excitability (reviewed in Refs. 5 and 6). In skeletal muscle, KATP channels are composed of pore-forming, Kir6.2, and regulatory, SUR2A, subunits.7 In the heart, sarcolemmal KATP channels have been shown to be in vivo composed of more proteins than SUR2A/Kir6.2, including creatine kinase (CK), muscle form of lactate dehydrogenase (M-LDH), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).8-12 It is yet unknown whether HOPP affects the level of fully assembled sarcolemmal KATP channels.

In the present study, we have taken advantage of skeletal muscle tissue from HOPP and gender/age-matched control patients to find out whether HOPP alters sarcolemmal KATP channels in any way. We have determined that the levels of sarcolemmal KATP channels are lower in a HOPP patient and this happens to be due to altered expression of the pore-forming Kir6.2 subunit.

Patients and Methods

Patients and samples

Skeletal muscle tissue samples (musculus brachioradialis) were taken from a HOPP patient and control patient with no history of muscle weakness undergoing a surgical reparative procedure following trauma to the limb (both patients were subjected to similar surgical procedures unrelated to HOPP). The HOPP patient was a male 45 years old who had been having profound attacks of weakness since the age of 7 years and during the attacks hypokalemia was observed (K+ was measured to be 2.7 mol/L). A typical HOPP-causing mutation, substitution of an arginine by a histidine residue at position 528 of the CACLN1A3 gene, has been found in this patient. On the other hand, a control patient was age and sex matched to the HOPP patient and did not suffer from any muskulo-skeletal disease. Samples of skeletal muscle were cut in several pieces and emerged in liquid nitrogen to be used for immunoprecipitation/Western blotting and real-time RT-PCR analysis.

Immunoprecipitation/Western blotting

Sheep anti-SUR2 and anti-Kir6.2 antibodies were used for immunoprecipitation and Western blotting in this study (described in detail in Refs. 9, 10, and 12). To obtain the sarcolemmal fraction, skeletal muscle samples were homogenized in buffer I (TRIS 10 mM, NaH2PO4 20 mM, EDTA 1 mM, PMSF 0.1 mM, pepstatin 10 μ/mL, leupeptin 10 μg/mL, at pH = 7.8) and incubated for 20 minutes (at 4 °C). The osmolarity was restored with KCl, NaCl, and sucrose, and the obtained mixture was centrifuged at 500 × g. The supernatant was diluted in buffer II (imidazole 30 mM, KCl 120 mM, NaCl 30 mM, NaH2PO4 20 mM, sucrose 250 mM, pepstatin 10 μg/mL, leupeptin 10 μg/mL, at pH = 6.8) and centrifuged at 7000 × g, the pellet removed and supernatant centrifuged at 30,000 × g. The obtained pellet contains the sarcolemma fraction. Protein concentration was determined using the Bradford method. 10 μg of the epitope-specific Kir6.2 antibody or 40 μg of the epitope-specific SUR2A antibody was pre-bound to Protein-G Sepharose beads and used to immunoprecipitate from 50 μg of membrane fraction protein extract. The pellets of this precipitation were run on SDS polyacrylamide gels for Western analysis. Western blot probing was performed using 1/200 and 1/300 dilutions of anti-SUR2 and anti-Kir6.2 antibody, respectively, and detection was achieved using Protein-G HRP and ECL reagents. The band intensities were analyzed using the Quantiscan software (Biosoft, Cambridge, UK).

Real-time RT-PCR

Total RNA was extracted from skeletal muscle tissue of transgenic using TRIZOL reagent (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Extracted RNA was further purified with RNeasy Mini Kit (Qiagen, Crawley, UK). The specific primers for human SUR2A, Kir6.2, adenylate kinase type (AK), creatine kinase (CK), muscle form of lactate dehydrogenase (M-LDH), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUR2B, SUR1, and Kir6.1 were designed as depicted in Table 1. The reverse transcription (RT) reaction was carried out with ImProm-II Reverse Transcriptase (Promega, Southampton, UK). A final volume of 20 μL of RT reaction containing 4 μL of 5× buffer, 3 mM MgCl2, 20 U of RNasin® Ribonuclease inhibitor, 1 U of ImProm-II reverse transcriptase, 0.5 mM each of dATP, dCTP, dGTP, and dTTP, 0.5 μg of oligo(dT), and 1 μg of RNA was incubated at 42°C for 1 hour and then inactivated at 70°C for 15 minutes. The resulting cDNA was used as a template for real-time PCR. A SYBR Green I system was used for the RT-PCR and the 25 μL reaction mixture contained: 12.5 μL of iQ™ SYBR® Green Supermix (2×), 7.5 nM each primers, 9 μL of ddH2O, and 2 μL of cDNA. In principle, the thermal cycling conditions were as follows: an initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 10 seconds of denaturing at 95°C, 15 seconds of annealing at 56°C, and 30 seconds of extension at 72°C. This protocol was modified for the m-LDH-, GAPDH-, and CK-specific primers changing the extension temperature to 55°C. The real-time PCR was performed in the same wells of a 96-well plate in the iCycler iQ™ Multicolor Real-Time Detection System (BioRad, Hercules, CA, USA). Data were collected following each cycle and displayed graphically (iCycler iQ™ Real-time Detection System Software, version 3.0A, BioRad). Primers were tested for their ability to produce no signal in negative controls by dimer formation and then with regard to the efficiency of the PCR reaction. Efficiency is evaluated by the slope of the regression curve obtained with several dilutions of the cDNA template. Melting curve analysis tested the specificity of primers. Threshold cycle values, PCR efficiency (examined by serially diluting the template cDNA and performing PCR under these conditions), and PCR specificity (by constructing the melting curve) were determined by the same software. Each cDNA sample was duplicated; the corresponding no-RT mRNA sample was included as a negative control (blank).

Table 1
Specific primers for known KATP channel-forming proteins


Kir6.2 and SUR2A physically associate with each other to form sarcolemmal KATP channel.7 Gene sequencing has demonstrated that Kir6.2 and SUR2 genes were not mutated by HOPP (data not shown). We have previously shown that immunoprecipitation with antibody raised against one subunit and probing the precipitate with the antibody raised against the other subunit provide an accurate measure of number of fully assembled and functional KATP channels.13,14 Here, immunoprecipitation with anti-SUR2A antibody and Western blotting with anti-Kir6.2 antibody has shown that Kir6.2 physically associated with SUR2A is present in less amounts in sarcolemma of HOPP than in those in the control patient (Figure 1; intensity of Kir6.2 signal was 69 AU and 33 AU in control and HOPP patient, respectively). Similar results were obtained when anti-Kir6.2 antibody was used for immunoprecipitation and anti-SUR2 antibody for Western blotting (Figure 1; intensity of SUR2A signal was 43 AU and 16 AU in control and HOPP patient, respectively).

Figure 1
Skeletal muscle of HOPP patient is deficient in KATP channels. Western blot with anti-Kir6.2 and anti-SUR2A antibody and corresponding graphs of anti-SUR2A and anti-Kir6.2 immunoprecipitate (IP) from sarcolemmal skeletal muscle fraction obtained from ...

In the heart, it has been shown that the changes in level of fully assembled sarcolemmal KATP channels are not associated with changes in expression of all components of the KATP channel protein complex. In fact, it has been demonstrated that fluctuations in expression of SUR2A alone are sufficient to affect the number of cardiac sarcolemmal KATP channels.13-17 To determine the underlying cause of decrease in numbers of skeletal muscle sarcolemmal KATP channels in a HOPP patient, we have measured mRNA levels of known KATP channel-forming proteins, including those that probably do not make KATP channels in skeletal muscle tissue. There was no difference between control and HOPP patients in expression of SUR2A, SUR2B, SUR1, AK, CK, GAPDH, and M-LDH (Figure S1). A single cycle of difference between two patients in Kir6.1 subunit has been observed (22.9 for control and 24.0 for HOPP patient; Figure S1). When Kir6.2 mRNA levels were measured, three cycles of differences were detected between control and HOPP patient (24.4 for control and 27.4 for HOPP patient; Figure S1), which would be equivalent to approximately 15 times difference.


Consistent with the nature of the clinical abnormalities in HOPP, this study has shown that a patient suffering from HOPP has altered expression of the Kir6.2 subunit of the skeletal muscle KATP channel.

It has been previously suggested that ion current through sarcolemmal KATP channels in skeletal muscle of HOPP patients is impaired.4 Here we have shown that the sequence of the main KATP channel-forming proteins, Kir6.2, and SUR2A subunits are unchanged in HOPP patients, suggesting that the mutation of KATP channel subunits is not a cause of any changes in KATP channels in HOPP patients.

The molecular structure of sarcolemmal KATP channels is still to be determined, but it has been shown that the electrophysiological properties correspond to those composed of Kir6.2 and SUR2A subunits.7 In the present study, immunoprecipitation followed by the Western blotting has shown that a HOPP patient is deficient in fully assembled sarcolemmal KATP channels. The function of KATP channels in skeletal muscle is still to be fully understood. In animal experimental models, skeletal KATP channels have been associated with the regulation of muscle fatigue. Opening of the skeletal KATP channels increases outward K+ currents that contribute to shortening of action potential duration, leading to protection against intracellular Ca2+ overload.18 Transgenic mice lacking functional KATP channels are characterized by increased muscle resting tension, impaired recovery of tetanic force after fatigue, and increased susceptibility of skeletal muscle fibers to physical exercise.2,3 In human skeletal muscle, KATP channels have been shown to serve as regulators of interstitial K+ concentration.19 Taken all together, manifestations of decreased number of KATP channels in skeletal muscle are similar to clinical signs of HOPP.

As the KATP channel is a multiprotein complex, it is yet to be fully understood how numbers of fully assembled KATP channels are controlled. It has been shown that the activity of SUR2 promoter alone regulates the overall number of KATP channels in the heart.13-17 In the present study, mRNA levels of SUR2A and SUR2B, which are both regulated by SUR2 promoter, did not differ between the two patients. This would exclude the possibility that the observed difference in number of assembled channels was due to impaired signaling that involves SUR2 promoter. In fact, out of all known KATP channel-forming proteins, the expression of Kir6.2 was the only one that was clearly affected. Kir6.2 is a pore-forming subunit.7 In the heart, it has been shown that Kir6.2 is expressed in vast excess to SUR2A (real-time RT-PCR threshold cycle was approximately 22 for Kir6.2 and 28.5 for SUR2A, which is a huge difference in the level of expression of these two subunits, 17), which seems to result in SUR2A being the main controller of the number of cardiac sarcolemmal KATP channels.13-17 However, as opposed to the heart, SUR2A is expressed in excess over Kir6.2 in the skeletal muscle (real-time RT-PCR threshold cycle was approximately 26.2 for Kir6.2 and 24.4 for SUR2A for the control skeletal muscle found in this study). As the least-expressed KATP channel-forming protein, Kir6.2 is probably a rate-limiting factor in making fully composed KATP channels. Our finding that down-regulation of Kir6.2 is a cause of altered number of KATP channels in skeletal muscle in HOPP patients supports this hypothesis.


In conclusion, this study has shown that a patient suffering from HOPP has impaired expression of the Kir6.2 subunit of the skeletal muscle KATP channel. This results in a deficiency in skeletal muscle KATP channels, which could explain symptoms and clinical signs of HOPP.


This study was supported by grants from BBSRC, British Heart Foundation, MRC, TENOVUS-Scotland, Wellcome Trust, and Anonymous Trust.


Supplemental Material

The following supplementary material is available for this article online:

Figure S1. A series of graphs illustrating real-time RT-PCR standard curves, melting curves and progress curves with corresponding graphs for known KATP channel forming proteins.

This material is available as part of the online article from


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