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?-Catenin Controls the Electrophysiologic Properties of Skeletal Muscle Cells by Regulating the ?2 Isoform of Na⁺/K⁺-ATPase.

ABSTRACT: ?-Catenin is a key component of the canonical Wnt signaling pathway. It has been shown to have an important role in formation of the neuromuscular junction. Our previous studies showed that in the absence of ?-catenin, the resting membrane potential (RMP) is depolarized in muscle cells and expression of the ?2 subunit of sodium/potassium adenosine triphosphatase (?2NKA) is reduced. To understand the underlying mechanisms, we investigated the electrophysiologic properties of a primary cell line derived from mouse myoblasts (C2C12 cells) that were transfected with small-interfering RNAs and over-expressed plasmids targeting ?-catenin. We found that the RMP was depolarized in ?-catenin knocked-down C2C12 cells and was unchanged in ?-catenin over-expressed muscle cells. An action potential (AP) was not released by knockdown or over-expression of ?-catenin. ?2NKA expression was reduced by ?-catenin knockdown, and increased by ?-catenin over-expression. We showed that ?-catenin could interact physically with ?2NKA (but not with ?1NKA) in muscle cells. NKA activity and ?2NKA content in the cell membranes of skeletal muscle cells were modulated positively by ?-catenin. These results suggested that ?-catenin (at least in part) regulates the RMP and AP in muscle cells, and does so by regulating ?2NKA.

SUBMITTER: Zhao C

PROVIDER: S-EPMC6693565 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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β-Catenin Controls the Electrophysiologic Properties of Skeletal Muscle Cells by Regulating the α2 Isoform of Na<sup>+</sup>/K<sup>+</sup>-ATPase.

Zhao Congying C Yu Yonglin Y Zhang Yi Y Shen Jue J Jiang Lihua L Sheng Guoxia G Zhang Weiqin W Xu Lu L Jiang Kewen K Mao Shanshan S Jiang Peifang P Gao Feng F

Frontiers in neuroscience 20190807

β-Catenin is a key component of the canonical Wnt signaling pathway. It has been shown to have an important role in formation of the neuromuscular junction. Our previous studies showed that in the absence of β-catenin, the resting membrane potential (RMP) is depolarized in muscle cells and expression of the α2 subunit of sodium/potassium adenosine triphosphatase (α2NKA) is reduced. To understand the underlying mechanisms, we investigated the electrophysiologic properties of a primary cell line d ...[more]

PMID: 31440132

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Project description:The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756-765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639-641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase alpha2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase alpha2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase alpha subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.

Project description:Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

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?-Catenin Controls the Electrophysiologic Properties of Skeletal Muscle Cells by Regulating the ?2 Isoform of Na+/K+-ATPase.

Publications

β-Catenin Controls the Electrophysiologic Properties of Skeletal Muscle Cells by Regulating the α2 Isoform of Na<sup>+</sup>/K<sup>+</sup>-ATPase.

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?-Catenin Controls the Electrophysiologic Properties of Skeletal Muscle Cells by Regulating the ?2 Isoform of Na⁺/K⁺-ATPase.