| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Our previous studies have identified several functional domains in the Na/K-ATPase alpha subunit ; (i)the ouabain-binding region (Ala70-Asp200), (ii)Na^+-sensitive segment (Met1-Leu69), (iii)K^+-sensitive segments (Phe920-Tyr1021), and (iv)subunit assembly domain (Asn894-Ala919). Under the current support (4/1996-3/1998), we first demonstrated that the Na^+-sensitive segment interact with the FSBA-binding region. Second, we showed that the calmodulin-binding auto-inhibitory domain (CBD) of the plasma membrane (PM) Ca-ATPase can regulate the Na/K-ATPase via direct interaction with the cytoplasmic FSBA-region of the alpha subunit. Namely, a chimeric protein (alpha-CBD), in which the carboxy terminal 165 amino acids (CBD) of the rat brain PM Ca-ATPase II were added to the carboxy terminus of the ouabain-sensitive chicken Na/K-ATPase alpha1 subunit, is an ouabain-sensitive Na/K-ATPase which can be activated by Ca^<2+> and calmodulin in a dose-dependent manner. Electrical properties of alph
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a-CBD characterized by two electrode voltage clamp indicated an altered ion-transport stoichiometry from 3Na^+, 2K^+ to 2Na^+, 2K^+. In addition, alpha-CBD was found to lack transient change movement in Na^+/Na^+ exchange mode. We conclude that the binding of the third Na^+ ion, but not the two others, in 3Na^+, 2K^+ transport mode apparently senses the electric field, and that the voltage-dependent Na^+-binding is likely to be lost in the chimera with CBD.Third, we also demonstrated that he SERCA-ATPase possesses two different population of K^+-binding sites, one of which could also bind Na^+. This population of K^+-/Na^+-binding sites possesses a higher affinity for K^+ than for Na^+. The K^+-/Na^+-binding site is localized within the putative transmembrane domains, M3, M4, M5, and M6. The putative transmembrane domains, M3, M4, M5, and M6, are suggested to be involved in the ion-dependent activation of P-type ATPase in general. In conclusion, the Na/K-ATPase (and also Ca-ATPase) consists of several domains with distinct functions, and that these domains can interact each other. Less
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