Structure-dynamic and functional relationships in a Li⁺-transporting sodium‑calcium exchanger mutant

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>

Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins.

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>

Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger

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>

TC-99m MDP SCINTIGRAPHIC EVALUATION OF STRESS FRACTURES OF THE FEET USING A 1 to 4 GRADING SYSTEM

Atlan, H.,Chisin, R.,Milgrom, C.,Giladi, M.,Stein, M.,Kashtan, H.,Margulies, J. 대한핵의학회 1984 핵의학 분자영상 Vol.18 No.1

Reassessment of Relative Value in Shoulder and Elbow Surgery: Do Payment and Relative Value Units Reflect Reality?

Suresh K Nayar,Richard L. Skolasky,Dawn M LaPorte,Ryan M Zimmerman,Aviram M Giladi,Umasuthan Srikumaran 대한정형외과학회 2021 Clinics in Orthopedic Surgery Vol.13 No.1

Background: Many U.S. health care institutions have adopted compensation models based on work relative value units (wRVUs) to standardize payments and incentivize providers. A major determinant of payment and wRVU assignments is operative time. We sought to determine whether differences in estimated operative times between the Centers for Medicare & Medicaid Services (CMS) and the National Surgical Quality Improvement Program (NSQIP) contribute to payment and wRVU misvaluation for the most common shoulder/elbow procedures. Methods: We collected data on wRVUs, payments, and operative times from CMS for 29 types of isolated arthroscopic and open shoulder/elbow procedures. Using regression analysis, we compared relationships between these variables, in addition to median operative times reported by NSQIP (2013–2016). We then determined the relative valuation of each procedure based on operative time. Results: Seventy-nine percent of CMS operative time were longer than NSQIP time (R2 = 0.58), including, but not limited to, shoulder arthroplasty and arthroscopic shoulder surgery. The correlation between payments and operative times was stronger between CMS data (R2 = 0.61) than NSQIP data (R2 = 0.43). Similarly, the correlation between wRVUs and operative times was stronger when using CMS data (R2 = 0.87) than NSQIP data (R2 = 0.69). Nearly all arthroscopic shoulder procedures (aside from synovectomy, debridement, and decompression) were highly valued according to both datasets. Per NSQIP, compensation for revision total shoulder arthroplasty ($10.14/min; 0.26 wRVU/min) was higher than that for primary cases ($9.85, 0.23 wRVU/min) and nearly twice the CMS rate for revision cases ($5.84/min; 0.13 wRVU/min). Conclusions: CMS may overestimate operative times compared to actual operative times as recorded by NSQIP. Shorter operative times may render certain procedures more highly valued than others. Case examples show that this can potentially affect patient care and incentivize higher compensating procedures per operative time when less-involved, shorter operations have similar patient-reported outcomes.