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RISS 인기검색어
- 검색키워드
- 저자 : Khananshvili, Daniel (검색결과 3 건)
- 무료
- 기관 내 무료
- 유료
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Lee, Su Youn,Giladi, Moshe,Bohbot, Hilla,Hiller, Reuben,Chung, Ka Young,Khananshvili, Daniel The Federation of American Societies for Experimen 2016 The FASEB Journal Vol.30 No.3
<P>Tissue-specific splice variants of Na+/Ca2+ exchangers contain 2 Ca2+-binding regulatory domains (CBDs), CBD1 and CBD2. Ca2+ interaction with CBD1 activates sodium-calcium exchangers (NCXs), and Ca2+ binding to CBD2 alleviates Na+-dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C-F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2-domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen-deuterium exchange mass spectrometry and stopped-flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprotein, which primarily predefines the extent of responses to Ca2+ binding. Deuterium uptake levels were up to 20% lower in the cardiac vs. the brain CBD12, reveling that elongation of the CBD2 FG loop by cassette exons rigidifies the interdomain Ca2+ salt bridge at the 2-domain interface, which secondarily modulates the Ca2+-bound states. In matching splice variants, the extent of Ca2+-induced rigidification correlates with decreased (up to 10-fold) Ca2+ off rates, where the cardiac CBD12 exhibits the slowest Ca2+ off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca2+ binding sites, which may serve as a structure-dynamic basis for splicing-dependent regulation of NCX.</P>
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Giladi, Moshe,Lee, Su Youn,Refaeli, Bosmat,Hiller, Reuben,Chung, Ka Young,Khananshvili, Daniel Elsevier 2019 Biochimica et biophysica acta, Bioenergetics Vol.1860 No.3
<P><B>Abstract</B></P> <P>The cell membrane (NCX) and mitochondrial (NCLX) Na<SUP>+</SUP>/Ca<SUP>2+</SUP> exchangers control Ca<SUP>2+</SUP> homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na<SUP>+</SUP> and Ca<SUP>2+</SUP>, whereas NCLX can exchange Ca<SUP>2+</SUP> with either Na<SUP>+</SUP> or Li<SUP>+</SUP>. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (K<SUB>int</SUB> = 0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (K<SUB>int</SUB> = 0.1–0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the “Ca<SUP>2+</SUP>-site” (S<SUB>Ca</SUB>) of NCLX_Mj binds Na<SUP>+</SUP>, Li<SUP>+</SUP>, or Ca<SUP>2+</SUP>, whereas one or more additional Na<SUP>+</SUP>/Li<SUP>+</SUP> sites of NCLX_Mj are incompatible with the Na<SUP>+</SUP> sites (S<SUB>ext</SUB> and S<SUB>int</SUB>) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na<SUP>+</SUP>/Li<SUP>+</SUP> (but not for Ca<SUP>2+</SUP>) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca<SUP>2+</SUP> homeostasis in distinct sub-cellular compartments.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Distinct structural elements control ion selectivity in the Na<SUP>+</SUP>/Ca<SUP>2+</SUP> exchanger. </LI> <LI> Residue-based changes in ion-selectivity specifically alter conformational patterns. </LI> <LI> Structure-dynamic variances at distinct sites are reflected in functional diversity. </LI> <LI> Conformational dynamics of apo-protein predefines a degree of functional asymmetry. </LI> <LI> The loss in ion selectivity has relatively small effects on the transport rates. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
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Giladi, Moshe,Lee, Su Youn,Hiller, Reuben,Chung, Ka Young,Khananshvili, Daniel Biochemical Society 2015 The Biochemical journal Vol.465 No.3
<P>The Ca(2+)-dependent allosteric regulation of Na(+)/Ca(2+) exchanger (NCX) proteins represents Ca(2+) interaction with the cytosolic domains, CBD1 (calcium-binding domain 1) and CBD2, which is associated either with activation, inhibition or no response to regulatory Ca(2+) in a given splice variant. CBD1 contains a high affinity Ca(2+)-sensor (which is highly conserved among splice variants), whereas primary information upon Ca(2+) binding to CBD1 is modified by alternative splicing of CBD2, yielding the diverse regulatory responses to Ca(2+). To resolve the structure-dynamic determinants of splicing-dependent regulation, we tested two-domain tandem (CBD12) constructs possessing either positive, negative or no response to Ca(2+) using hydrogen-deuterium exchange MS (HDX-MS), SAXS, equilibrium 45Ca(2+) binding and stopped-flow kinetics. Taken together with previously resolved crystallographic structures of CBD12, the data revealed that Ca(2+) binding to CBD1 rigidifies the main-chain flexibility of CBD2 (but not of CBD1), whereas CBD2 stabilizes the apo-CBD1. Strikingly, the extent and strength of Ca(2+)-dependent rigidification of CBD2 is splice-variant dependent, where the main-chain rigidification spans from the Ca(2+)-binding sites of CBD1, through a helix of CBD2 (positioned at the domains' interface) up to the tip of CBD2 [>50 ? (1 ? = 0.1 nm)] or alternatively, it stops at the CBD2 helix in the splice variant exhibiting an inhibitory response to regulatory Ca(2+). These results provide a structure-dynamic basis by which alternative splicing diversifies the regulatory responses to Ca(2+) as well as controls the extent and strength of allosteric signal propagation over long distance.</P>
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