title: Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger.
abstract: Tissue-specific splice variants of Na(+)/Ca(2+) exchangers contain 2 Ca(2+)-binding regulatory domains (CBDs), CBD1 and CBD2. Ca(2+) interaction with CBD1 activates sodium-calcium exchangers (NCXs), and Ca(2+) 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 Ca(2+) 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 Ca(2+) salt bridge at the 2-domain interface, which secondarily modulates the Ca(2+)-bound states. In matching splice variants, the extent of Ca(2+)-induced rigidification correlates with decreased (up to 10-fold) Ca(2+) off rates, where the cardiac CBD12 exhibits the slowest Ca(2+) off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca(2+) binding sites, which may serve as a structure-dynamic basis for splicing-dependent regulation of NCX.
authors.affiliation: *School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon, South Korea; and Department of Physiology and Pharmacology, Tel-Aviv University, Tel-Aviv, Israel.
authors.forename: Su Youn
authors.initials: SY
authors.lastname: Lee
authors.affiliation: *School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon, South Korea; and Department of Physiology and Pharmacology, Tel-Aviv University, Tel-Aviv, Israel.
authors.forename: Moshe
authors.initials: M
authors.lastname: Giladi
authors.affiliation: *School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon, South Korea; and Department of Physiology and Pharmacology, Tel-Aviv University, Tel-Aviv, Israel.
authors.forename: Hilla
authors.initials: H
authors.lastname: Bohbot
authors.affiliation: *School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon, South Korea; and Department of Physiology and Pharmacology, Tel-Aviv University, Tel-Aviv, Israel.
authors.forename: Reuben
authors.initials: R
authors.lastname: Hiller
authors.affiliation: *School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon, South Korea; and Department of Physiology and Pharmacology, Tel-Aviv University, Tel-Aviv, Israel kychung2@skku.edu dhanan@post.tau.ac.il.
authors.forename: Ka Young
authors.initials: KY
authors.lastname: Chung
authors.affiliation: *School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon, South Korea; and Department of Physiology and Pharmacology, Tel-Aviv University, Tel-Aviv, Israel kychung2@skku.edu dhanan@post.tau.ac.il.
authors.forename: Daniel
authors.initials: D
authors.lastname: Khananshvili
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics; D041961:Deuterium Exchange Measurement / Q000379:methods; D004285:Dogs; D005091:Exons / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics*; D017136:Ion Transport / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000235:genetics*
chemical_list: D002135:Calcium-Binding Proteins; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium
keywords: HDX-MS; alternative splicing; conformational dynamics; intrinsically disordered proteins