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

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.