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Abstract
Changes in intracellular Ca2+ concentrations ([Ca2+]i) are an important signal for various physiological activities. The Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate [Ca2+]i homeostasis. Calmodulin (CaM) senses [Ca2+]i changes and relays Ca2+ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM1234 (a Ca2+-binding deficient mutant) in HEK293T cells and measured the increase in [Ca2+]i contributed by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM1234. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.
Highlights
The change in the intracellular Ca2+ concentration ([Ca2+]i) is an important signal that controls versatile cellular processes, and there are several mechanisms that maintain Ca2+ homeostasis
Aligning bovine NCX1 with the conserved CaM-binding 1-5-8-14 motif identified in calcineurin [22], the plasma membrane Ca2+-ATPase [17], L-type Ca2+ channel [23], and the small conductance Ca2+-activated K+ channel [24] shows a conserved 1-5-8-14 segment in NCX1 (CaMS, a.a. residues 716– 735) (Fig 1B) positioned after the alternative splicing region
The other two Na+/Ca2+ exchangers (NCX) isoforms, NCX2 and NCX3, contain this putative 1-5-8-14 motif at the C-terminus of the large cytosolic loop; no predicted CaM binding motif is present in Na+/Ca2+-K+ exchanger (NCKX) isoforms (Calmodulation database and meta-analysis target site predictor, http://cam.umassmed.edu/)
Summary
The change in the intracellular Ca2+ concentration ([Ca2+]i) is an important signal that controls versatile cellular processes, and there are several mechanisms that maintain Ca2+ homeostasis. Two different families of solute carriers are responsible for Na+ gradient-dependent Ca2+ transport: SLC8, a Na+/Ca2+ exchanger (NCX) with a stoichiometry of 3 Na+ in exchange for 1 Ca2+, and SLC24, a Na+/Ca2+-K+ exchanger (NCKX) with a stoichiometry of 4 Na+ in exchange for 1 Ca2+ and 1 K+ [2,4,5,6]. NCX1 is composed of 970 a.a. residues and is predicted to have 10 transmembrane segments, with a large intracellular loop between the 5th and 6th transmembrane segments (TMs) (Fig 1A). At the N-terminal of the intracellular loop, an XIP (exchanger inhibitory peptide) segment attenuates the Na+-dependent inactivation of NCX1 [10]. This is followed by a catenin-like domain whose function is not yet clear. The C-terminal of CBD2 contains an alternative splicing region that determines the stability of CBD2 [14]
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