Targeted mutation of mouse skeletal muscle sodium channel produces myotonia and potassium-sensitive weakness
Lawrence J. Hayward, Joanna S. Kim, Ming-Yang Lee, Hongru Zhou, Ji W. Kim, Kumudini Misra, Mohammad Salajegheh, Fen-fen Wu, Chie Matsuda, Valerie Reid, Didier Cros, Eric P. Hoffman, Jean-Marc Renaud, Stephen C. Cannon, Robert H. Brown Jr.
Lawrence J. Hayward, Joanna S. Kim, Ming-Yang Lee, Hongru Zhou, Ji W. Kim, Kumudini Misra, Mohammad Salajegheh, Fen-fen Wu, Chie Matsuda, Valerie Reid, Didier Cros, Eric P. Hoffman, Jean-Marc Renaud, Stephen C. Cannon, Robert H. Brown Jr.
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Research Article Muscle biology

Targeted mutation of mouse skeletal muscle sodium channel produces myotonia and potassium-sensitive weakness

Abstract

Hyperkalemic periodic paralysis (HyperKPP) produces myotonia and attacks of muscle weakness triggered by rest after exercise or by K+ ingestion. We introduced a missense substitution corresponding to a human familial HyperKPP mutation (Met1592Val) into the mouse gene encoding the skeletal muscle voltage-gated Na+ channel NaV1.4. Mice heterozygous for this mutation exhibited prominent myotonia at rest and muscle fiber-type switching to a more oxidative phenotype compared with controls. Isolated mutant extensor digitorum longus muscles were abnormally sensitive to the Na+/K+ pump inhibitor ouabain and exhibited age-dependent changes, including delayed relaxation and altered generation of tetanic force. Moreover, rapid and sustained weakness of isolated mutant muscles was induced when the extracellular K+ concentration was increased from 4 mM to 10 mM, a level observed in the muscle interstitium of humans during exercise. Mutant muscle recovered from stimulation-induced fatigue more slowly than did control muscle, and the extent of recovery was decreased in the presence of high extracellular K+ levels. These findings demonstrate that expression of the Met1592Val Na+ channel in mouse muscle is sufficient to produce important features of HyperKPP, including myotonia, K+-sensitive paralysis, and susceptibility to delayed weakness during recovery from fatigue.

Authors

Lawrence J. Hayward, Joanna S. Kim, Ming-Yang Lee, Hongru Zhou, Ji W. Kim, Kumudini Misra, Mohammad Salajegheh, Fen-fen Wu, Chie Matsuda, Valerie Reid, Didier Cros, Eric P. Hoffman, Jean-Marc Renaud, Stephen C. Cannon, Robert H. Brown Jr.

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Figure 1

Knock-in of human Met1592Val variant into mouse NaV1.4 gene.

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Knock-in of human Met1592Val variant into mouse NaV1.4 gene. ...
(A) Na+ channel topology and the location of the Met1592Val substitution within the S6 segment of domain IV (top). The mNaV1.4 targeting sequence (bottom) encompassed exons 17–24 (black boxes) and the 3′-UTR of exon 24 (hatched area), while the PGKneo selection gene was inserted within intron 23. The Met→Val mutation (*) and the silent HpaI site (H) are indicated within exon 24. Bold X’s indicate the limits of the targeting sequence. Shown are the internal and external hybridization probe regions and restriction enzyme sites for AvrII (A), AflII (A2), KpnI (K), NheI (N), EcoRI (R), SacI (S), and ScaI (Sc). (B) Southern blot of NheI-digested mouse genomic DNA (10 μg per lane) hybridized with the internal 3′-UTR probe demonstrates a 4.5-kb wild-type allele or a 6.1-kb allele produced by insertion of the PGKneo gene. Genotype is indicated for wild-type (+/+), heterozygous mutant (+/m), and homozygous mutant (m/m) alleles. (C) Southern blot of DNA digested with AflII plus HpaI plus ScaI and hybridized to the external probe identifies an 8.0-kb wild-type allele (ScaI–AflII fragment) and a 4.4 kb mutant allele (HpaI–AflII fragment). (D) Genotyping by analysis of a 1.39-kb PCR product amplified from exon 24 using primers a and b (see Supplemental Methods). The mutant allele contained a novel HpaI site and lacked one of the 5 NspI sites present in the wild-type allele (indicated by thick vertical lines under the asterisk, with sequence shown below). The lane labeled “M” shows a 100-bp marker DNA ladder.