Structure-dynamic coupling through Ca2+-binding regulatory domains of mammalian NCX isoform/splice variants

Abstract

Mammalian Na+/Ca2+ exchangers (NCX1, NCX2, and NCX3) and their splice variants are expressed in a tissue-specific manner and are regulated by Ca2+ binding CBD1 and CBD2 domains. NCX2 does not undergo splicing, whereas in NCX1 and NCX3, the splicing segment (with mutually exclusive and cassette exons) is located in CBD2. Ca2+ binding to CBD1 results in Ca2+-dependent tethering of CBDs through the network of interdomain salt-bridges, which is associated with NCX activation, whereas a slow dissociation of “occluded” Ca2+ inactivates NCX. Although NCX variants share a common structural basis for Ca2+-dependent tethering of CBDs, the Ca2+ off-rates of occluded Ca2+ vary up to 50-fold, depending on the exons assembly. The Ca2+-dependent tethering of CBDs rigidifies the interdomain movements of CBDs without any significant changes in the CBDs’ alignment; consequently, more constraining conformational states become more populated in the absence of global conformational changes. Although this Ca2+-dependent “population shift” is a common mechanism among NCX variants, the strength and span of backbone rigidification from the C-terminal of CBD1 to the C-terminal of CBD2 is exon dependent. The mutually exclusive exons differentially stabilize/destabilize the backbone dynamics of Ca2+-bound CBDs in NCX1 and NCX3 variants, whereas the cassette exons control the stability of the interdomain linker. The combined effects of mutually exclusive and cassette exons permit a fine adjustment of two different regulatory pathways: the Ca2+-dependent activation (controlled by CBD1) and the Ca2+-dependent alleviation of Na+-induced inactivation (controlled by CBD2). Exon-controlled dynamic features match with cell-specific regulatory requirements in a given variant.