Alternative Splicing at CBD2 Domain Modifies Ca2+ Sensing Properties at CBD1 in Regulatory Two-Domain Tandem of NCX Proteins

Abstract

The Na+/Ca2+ exchanger isoforms (NCX1-3) and their splice variants are expressed in a tissue-specific manner and extrude Ca2+ from the cell to regulate Ca2+-dependent events in many cell-types. The cytosolic f-loop of NCX proteins consists of two regulatory domains, CBD1 and CBD2, which form a two-domain tandem (CBD12) with a short linker. The CBD1 domain contains two high-affinity Ca2+ sites (Ca3-Ca4), which largely determine Ca2+-dependent allosteric activation of NCX, whereas the alternative splicing sequence is exclusively located on CBD2. Previous structural, biochemical and mutational studies have shown that in an isolated CBD12 tandem, CBD2 interacts with CBD1 through a network of interdomain salt-bridges to modify Ca2+ affinity and off-rates at high affinity Ca2+ sensor of CBD1. We posit here that splice variations of A-F exons on CBD2 specifically modify dynamic properties of Ca2+ sensing at CBD1. In this respect, we analyzed three splice variants of an isolated two-domain construct, NCX1-CBD12: kidney (BD), brain (AD) and cardiac (ACDEF). In addition, we have analyzed CBDs from CALX-CBD12, a Drosophila NCX protein which is inactivated, rather than activated by Ca2+ binding to the high affinity sensor on CBD1. By using equilibrium binding, stopped-flow and SAXS (small-angle X-ray scattering), we demonstrate that different splice variants of CBD2 specifically modify the kinetic and equilibrium properties of Ca2+ sensing at CBD1. Moreover, Ca2+ binding decreases maximum distance (Dmax) of the NCX-CBD12 kidney, brain and cardiac splice variants, whereas Ca2+ has the opposite effect on CALX-CBD12. Therefore, experimental approaches described here may help in identifying specific structural motifs in CBD2, responsible for the dynamic properties of Ca2+ sensing and also the mode of allosteric signal decoding.