Department of Molecular Physiology

Members

Director: 

Shigeo Wakabayashi 

Laboratory chiefs: 

Yuko Iwata, Tomoe Nishitani（working name, T. Y. Nakamura）

Research staff:

Takashi Hisamitsu, Hironori Hanada

Postdoctoral fellow:

Soushi Kobayashi

Trainees:

Yuko Kobayashi

Research Assistants:

Hitomi Ohtake, Miyuki Yuno, Kazuyo Endo

Research activities

The Department of Molecular Physiology has been engaged in studies of the molecular mechanisms of the ionic regulation of contraction and relaxation of cardiac and smooth muscles, which are the major constituents of the cardiovascular system. In particular, we focus on the molecular physiology and pharmacology of the sarcolemmal ion transporters. Another subject is a study of the cellular mechanism underlying the functional failure and death of myocytes associated with cardiomyopathy. This study is particularly concerned with the role of cytoskeletal proteins in the genesis of dilated cardiomyopathy using animal disease models.

(A) Studies on cellular ionic mechanisms regulating the contractile state of myocytes

(B) Studies on roles of cytoskeletal proteins in the genesis of cardiomyopathy

(C) Resent Publications

 

(A) Studies on cellular ionic mechanisms regulating the contractile state of myocytes

Ionic homeostasis in the cardiovascular system is regulated by multiple ion transporters and ion channels. Among them, the plasma membrane Na⁺/Ca²⁺ (NCX) and Na⁺/H⁺ (NHE) exchangers play crucial roles in ionic metabolism of intracellular Na⁺, Ca²⁺ and H+ in cardiovascular cells. These transporters participate not only in physiological regulation of contractile activity but also in pathological aspects of myocyte function such as cardiac ischemia/reperfusion injury, cardiac hypertrophy/failure, and hypertension. In this laboratory, we investigate various mechanistic, physiological, and pharmacological aspects of functions of these transporters, in particular, ion transport mechanisms, interaction with signaling molecules, and functions in cardiovascular tissues, using multiple methodologies including analysis of expressed mutant molecules, biochemical and cell biological approaches, and analysis using genetically modified mice.

We have developed the first selective inhibitor of NCX, KB-R7943, that has therapeutic potential for clinical use. This compound is being widely used as a useful tool for studies of NCX function at different levels of experimental systems. Recently, we analyzed the topology of the cardiac NCX molecule as well as its interaction with transport substrate Ca²⁺, KB-R7943, a nonselective inhibitor Ni²⁺, and an activator Li+. Based on detailed analysis by cysteine scanning and site-directed mutagenesis, we found that the highly conserved alpha-1 and alpha-2 repeat regions in NCX may be involved in the interactions with these inhibitors and ligands, suggesting that these regions participate in the formation of ion transport pathway in NCX. Our study have also obtained evidence that NCX consists of 9 transmembrane segments, with regions containing alpha-1 and alpha-2 repeats forming re-entrant membrane loops originating from the opposite sides of the membrane.

NHE is an excellent target for the study of cellular signal transduction leading to modulation of transport activity, because it is regulated by a variety of extracellular signals. We have recently analyzed the interaction of calmodulin (CaM) and the calcineurin-homologous protein CHP with various NHE isoforms, and found that CaM is involved in Ca2+-induced activation of NHE1, whereas CHP is an essential cofactor to support physiological activity of multiple NHE isoforms. We have also determined that a hallmark of NHE regulation "pH-sensor" is regulated via several cytoplasmic subdomains of NHE1. More recently, we analyzed membrane topology of NHE1, and found that NHE1 comprises 12 transmembrane segments with N- and C-termini in the cytosol. Our future goal is to understand the structure-function relationships in these ion transporters and its regulatory machinery at a higher resolution as well as to obtain insights into their definitive physiological roles in cardiovascular and other tissues.

(B) Studies on roles of cytoskeletal proteins in the genesis of cardiomyopathy

Cardiomyopathy is caused by a variety of etiologies. Deficiency in the dystrophin gene causes a high incidence of dilated cardiomyopathy in Duchenne and Becker muscular dystrophy patients and also causes X-linked dilated cardiomyopathy. In animal models, dilated cardiomyopathies have been associated with genetic deficiencies of several cytoskeletal proteins, indicating that these proteins are essential for the maintenance of the normal contractile function of cardiac myocytes. However, the cellular mechanisms by which particular defects in these proteins generate cardiomyopathy are not known.

In striated muscles, the dystrophin complex consists of dystrophin and its associated proteins: alpha- and beta-dystroglycans, alpha-, beta-, gamma- and delta-sarcoglycans, sarcospan, syntrophins, and dystrobrevins. The complex confers a linkage between laminin in the extracellular matrix and F-actin cytoskeleton via the alpha- and beta-dystroglycan subcomplex and is thought to protect sarcolemma from damage induced by contractile activity. Sarcoglycans are intrinsic membrane proteins and form a subcomplex with sarcospan. Loss-of-function mutations in sarcoglycan genes cause skeletal muscle dystrophy and cardiomyopathy in patients, BIO14.6 hamsters and sarcoglycan KO mice.

In skeletal and cardiac muscles of BIO14.6 hamsters, we found that sarcoglycans are greatly reduced or lost and that the dystrophin complex are totally disrupted, although dystrophin and beta-dystroglycan are present at mildly reduced levels. Using this hamster model, we have been studying the functions of sarcoglycans and the cellular mechanism for pathogenesis of cardiac and skeletal myopathies. We have recently provided evidence that sarcoglycan deficiency in BIO14.6 hamster myocytes or in myocytes rendered sarcoglycans deficient using sarcoglycan antisense oligonucleotides are susceptible to applied mechanical stress leading to cell damage and that these cells exhibit an increased basal rate of Ca²⁺ influx. In BIO14.6 hamster myocytes, we have further shown that the resting activity of stretch-activated non-specific cation channels is markedly elevated. The latter finding suggests that increased activity of these channels may ultimately cause Ca2+ overload, which contributes to cell damage observed in the myopathic myocytes. We have also studied the bidirectional signaling between sarcoglycan subcomplex and integrin cell adhesion systems in cultured L6 myocytes and the interaction of syntrophin with actin cytoskeleton in skeletal and cardiac myocytes. Furthermore, we are engaged in search of genes that are possibly involved in pathogenesis of cardiac and skeletal myopathies.

 

(C) Recent Publications

Hisamitsu, T., Ben Ammar, Y. Nakamura, T.Y., Wakabayashi, S.: Dimerization is crucial for the function of the Na⁺/H⁺ Exchanger NHE1. (2006) Biochemistry (in press).

Mishima, M., Wakabayashi, S., Kojima, C.: Solution structure of the cytoplasmic region of Na⁺/H⁺ exchanger 1 complexed with essential cofactor calcineurin B homologous protein 1. (2006) J. Biol. Chem. (in press).

Kobayashi, Y., Katanosaka, Y., Iwata, Y., Shigekawa, M, Wakabayashi, S.: Identification and characterization of GSRP-56, a novel Golgi-localized spectrin-repeat containing protein. (2006) Exp. Cell Res. 312: 3152-3164

Ben Ammar, Y., Takeda, S., Hisamitsu, T., Mori. H., Wakabayashi,S.: Crystal structure of CHP2 in complex with its binding region in NHE1 and insights into the mechanism of pH regulation. (2006) EMBO J. 25(11): 2315-2325

Nakamura, T.Y., Jeromin, A., Smith, G., Kurushima, H., Koga, H., Nakabeppu, Y., Wakabayashi, S., Nabekura, J.: Novel Role of Neuronal Ca²⁺ Sensor-1 as a Survival Factor Up-Refulated in Injured Neurons. (2006) J. Cell Biol. 172(7): 1081-1091

Ben Ammar, Y., Takeda, S., Sugawara, M., Miyano, M., Mori, H., Wakabayashi, S.: Crystallization and preliminary crystallographic analysis of the human calcineurin homologous protein CHP2 bound to the cytoplasmic region of the Na⁺/H⁺ exchanger NHE1. (2005) Acta Crystallographica Section F 61(10): 956-958

Iwata, Y., Katanosaka, Y., Zhu, S., Kobayashi, Y., Hanada, H., Shigekawa, M., Wakabayashi, S.: Protective effects of Ca²⁺ handling drugs against abnormal Ca²⁺ homeostasis and cell damage in myopathic skeletal muscle cells. (2005) Biochem. Pharmacol. 70: 740-751

Hamahata, K., Adachi, S., Matsubara, H., Okada, M., Imai, T., Watanabe, K., Toyokuni, S., Ueno, M., Wakabayashi, S., Katanosaka, Y., Akiba, S., Kubota, M., Nakahata, T.: Mitochondrial dysfunction is related to necrosis-like programmed cell death induced by A23187 in CEM cells. (2005) Eur. J. Pharmacol. 516: 187-196

Katanosaka, Y., Iwata, Y., Kobayashi, Y., Shibasaki, F., Wakabayashi, S., Shigekawa, M.: Calcineurin Inhibits Na⁺/Ca²⁺ Exchange in Phenylephrine-treated Hypertrophic Cardiomyocytes. (2005) J. Biol. Chem. 280(7): 5764-5772

Iwata, Y., Shigekawa, M., Wakabayashi, S.: Cardiac Syntrophin Isoforms: Species-Dependent Expression, Association with Dystrophin Complex and Subcellular Localization. (2005) Mol. Cell. Biochem. 268: 59-66

Iwata, Y., Sampaolesi, M., Shigekawa, M., Wakabayashi, S.: Syntrophin is an actin-binding protein the cellular localization of which is regulated through cytoskeletal reorganization in skeletal muscle cells. (2004) Eur. J. Cell Biol. 83: 555-565

Hisamitsu, T., Pang, T., Shigekawa, M., Wakabayashi, S.: Dimeric Interaction between the Cytoplasmic Domains of the Na⁺/H⁺ Exchanger NHE1 Revealed by Symmetrical Intermolecular Cross-Linking and Selective Co-Immunoprecipitation. (2004) Biochemistry 43(34): 11135-11143

Kokubo, Y., Inamoto, N., Tomoike, H., Kamide, K., Takiuchi, S., Kawano, Y., Tanaka, C., Katanosaka, Y., Wakabayashi, S., Shigekawa, M., Hishikawa, O., Miyata, T.: Association of Genetic Polymorphisms of Sodium-Calcium Exchanger 1 Gene, NCX1, with Hypertension in a Japanese General Population. (2004) Hypertension Res. 27(10): 697-702

Pang, T., Hisamitsu, T., Mori, H., Shigekawa, M., Wakabayashi, S.: Role of Calcineurin B Homologous Protein in pH Regulation by the Na⁺/H⁺ Exchanger 1: Tightly Bound Ca²⁺ Ions as Important Structural Elements. (2004) Biochemistry 43(12): 3628-3636

Muraki, K., Iwata, Y., Katanosaka, Y., Ito, T., Ohya, S., Shigekawa, M., Imaizumi, Y.: TRPV2 Is a Component of Osmotically Sensitive Cation Channels in Murine Aortic Myocytes. (2003) Circ. Res. 93: 829-838

Wakabayashi, S., Hisamitsu, T., Pang, T., Shigekawa, M.: Kinetic dissection of two distinct proton binding sites in Na⁺/H⁺ exchangers by measurement of reverse mode reaction. (2003) J. Biol. Chem. 278(44): 43580-43585

Iwata, Y., Katanosaka, Y., Arai, Y., Komamura, K., Miyatake, K., Shigekawa, M.: A novel mechanism of myocyte degeneration involving the Ca²⁺-permeable growth factor-regulated channel. (2003) J. Cell Biol. 161(5): 957-967

Wakabayashi, S., Hisamitsu, T., Pang, T., Shigekawa, M.: Mutations of Arg440 and Gly455/Gly456 Oppositely Change pH-Sensing of Na⁺/H⁺ Exchanger NHE1. (2003) J. Biol. Chem. 278(14): 11828-11835

Hirata, T., Kaneko, T., Ono, T., Nakazato, T., Furukawa, N., Hasegawa, S., Wakabayashi, S., Shigekawa, M., Chang, M.-H., Romero M., Hirose, S.: Mechanism of acid adaptation of a fish living in a pH 3.5 lake. (2003) Am J Physiol Regul Integr Comp Physiol. 284(5): R1199-R1212

Su, X., Pang, T., Wakabayashi, S., Shigekawa, M.: Evidence for involvement of the putative first extracellular loop in differential volume-sensitivity of the Na⁺/H⁺ exchangers NHE1 and NHE2. (2003) Biochemistry 42(4): 1086-1094

Pang, T., Wakabayashi, S., Shigekawa, M.: Expression of Calcineurin B Homologous Protein 2 Protects Serum Deprivation-Induced Cell Death by Serum-Independent Activation of Na⁺/H⁺ exchanger. (2002) J. Biol. Chem. 277(46): 43771-43777

Miyazaki, E., Sakaguchi, M., Wakabayashi, S., Shigekawa, M., Mihara, K.: NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell. (2001) J. Biol. Chem. 276(52): 49221-49227

Pang, T., Su, X., Wakabayashi, S., Shigekawa, M.: Calcineurin-homologous protein as an essential cofactor for Na⁺/H⁺ exchangers. (2001) J. Biol. Chem. 276(20): 17367-17372

Nakamura T.Y., Iwata Y., Sampaolesi, M., Hanada, H., Saito,N., Artman, M.,Coetzee, W.A., Shigekawa, M.: Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters. (2001) Am. J. Physiol. 281: C690-C699

Hisamitsu, T., Ohata, H., Kawanishi, T., Iwamoto, T., Shigekawa, M., Amano, H., Yamada, S., Momose, K.: A mechanism of Ca²⁺ release from Ca²⁺ stores coupling to the Na⁺/Ca²⁺ exchanger in cultured smooth muscle cells. (2001) Life Sci. 69: 2775-2787

Shigekawa, M., Iwamoto, T.: Cardiac Na⁺/Ca²⁺ Exchange. Molecular and Pharmacological Aspects. (2001) Circ. Res. 88: 864-876

Wakabayashi, S., Pang, T., Su, X., Shigekawa, M.: Structure-Function of the Na⁺/H⁺ exchangers. Role of interacting proteins. (2001) Kidney Int. 60: 403-404

Sampaolesi, M., Yoshida, T., Iwata, T., Hanada, H., Shigekawa, M.: Stretch-induced cell damage in sarcoglycan-deficient myotubes. (2001) Pflugers Arch. 442: 161-170

Wakabayashi, S., Pang, T., Su, X., Shigekawa, M.: Second mutations rescue point mutant of the Na+/H+ exchanger NHE1 showing defective surface expression. (2000) FEBS Lett. 487: 257-261

Iwamoto, T., Uehara, A., Imanaga, I., Shigekawa, M.: The Na⁺/Ca²⁺ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca²⁺ affinity. (2000) J. Biol. Chem. 275: 38571-38580

Wakabayashi, S., Pang, T., Su, X., Shigekawa, M.: A novel topology model of the human Na⁺/H⁺ exchanger isoform 1. (2000) J. Biol. Chem. 275: 7942-7949

Kobayashi, Y., Pang, T., Iwamoto, T., Wakabayashi, S., Shigekawa, M.: Lithium activates the Na⁺/H⁺ exchanger: Isoform specificity and inhibition by genistein. (2000) Pflugers Arch. 439: 455-462