SLC24A Family (K+-Dependent Na+-Ca2+ Exchanger, NCKX)
Tightly controlled changes in cytosolic free Ca2+ concentration are widely involved in cell signaling in most tissues of our body. Free Ca2+ concentration is increased through the activation of a wide range of surface or intracellular Ca2+ channels and is returned to resting values through the action of ATP-driven Ca2+ pumps located in the plasma membrane and endoplasmic reticulum as well as Na+/Ca2+ exchangers located on the plasma membrane. By the mid 1980s, it had been established that the surface membrane of the outer segments of retinal rod photoreceptors (ROS) contained a potent Na+-Ca2+ exchange mechanism (Schnetkamp 1980; Yau and Nakatani 1984), and, in 1988, the Na+-Ca2+ exchanger protein was purified from bovine ROS as a 220 kDa glycoprotein (Cook and Kaupp 1988). In 1989, it was discovered that Na+-Ca2+ exchange in ROS was a K+-dependent Na+/Ca2+ exchange process operating at a stoichiometry of four Na+ ions exchanged against one Ca2+ plus one K+ ion. This ion exchanger protein is now referred to as NCKX1 (Cervetto et al. 1989; Schnetkamp et al. 1989). In the early 1990s, the functional properties and physiological role of the NCKX1 exchanger in ROS were examined in significant detail as described below, and, to date, these remain the only detailed studies of NCKX function and physiology in native tissues. In 1992, the cDNA of the full-length bovine ROS NCKX was cloned and shown to encode a protein of 1,199 amino acids (Reiländer et al. 1992). In subsequent years, four additional and distinct human genes were identified encoding the NCKX2–5 proteins (Schnetkamp 2013). We now know that genes encoding NCKX proteins belong to the CaCA superfamily of cation/Ca2+ exchangers and their closest relatives in the superfamily are the Na+/Ca2+ exchanger (NCX) proteins carrying out K+-independent Na+/Ca2+ exchange and studied extensively in cardiomyocytes: the SLC24 gene family encodes the human NCKX1–5 proteins (Schnetkamp et al. 2014), while the SLC8 gene family encodes the human NCX1–3 proteins (Khananshvili 2013). NCKX1–5 proteins range between 500 and 750 residues with the exception of the mammalian NCKX1 proteins which can be as large as 1,200 residues. The increase in size is due to poorly conserved expansions of the two large hydrophilic loops that are present in all NCKX proteins (Szerencsei et al. 2002). NCKX and NCX proteins show very limited sequence homology restricted to two short segments of around 50 residues each. However, the topological arrangement of transmembrane α helices is now thought to be the same for both NCX and NCKX (Szerencsei et al. 2013). Initially, the SLC24 gene family was thought to contain a sixth member encoding NCKX6, but this is now designated as NCLX or Li+-permeable Na+-Ca2+ exchanger encoded by the SLC8B1 gene (Khananshvili 2013). In the next three sections, we will review the functional properties and physiological role of NCKX1 in phototransduction in ROS and then compare in situ functional properties with those obtained for NCKX1–4 proteins after heterologous expression in the human embryonic kidney cells (HEK293) cells. Next, we will discuss physiological roles for NCKX proteins as revealed by gene deletion studies and analysis of SLC24 mutations found in patients with congenital diseases, and, finally, we will review our current knowledge of structure-function relationships of NCKX as revealed by studies on wild-type (WT) NCKX2 and a large collection of mutant NCKX2 proteins expressed in cell lines.
Functional Properties and Physiological Roles
K+-dependent Na+/Ca2+ exchangers (NCKX) are bidirectional plasma membrane Ca2+ transporters that utilize the Na+ and K+ ionic gradients across the cell surface membrane and can remove Ca2+ from the cytoplasm or move Ca2+ into the cell, dependent on the prevailing Na+ and K+ gradients. NCKX proteins are thought to operate through the alternating access model of ion transport (Schnetkamp 2013, 2014), which is the accepted mode of transport for all primary and secondary transporters. This transport model implies that, within the transporter protein, the same binding pocket coordinates substrates when the transporter is in either the cytoplasmic configuration or the extracellular configuration. The alternating access model can be described with Michaelis-Menten kinetics, which defines an apparent substrate dissociation or Michaelis-Menten constant (Km) as a measure of substrate affinity. In the NCKX model, the cation-binding sites bind either Ca2+ and K+ or four Na+ ions, therefore suggesting that there exists a site for which Ca2+ and Na+ compete for binding and other sites where Na+ and K+ compete (Schnetkamp et al. 2014). However, transport only occurs with the occupancy of either one Ca2+ and one K+ or with the occupancy of four Na+ ions. Transport studies have shown that NCKX proteins are absolutely selective for Na+; however, Ca2+ and K+ ions can be replaced by Sr2+ and Rb+ (or NH4+), respectively. The ionic affinities of NCKX1–4 proteins have been recently reviewed (Schnetkamp et al. 2014; Jalloul et al. 2016b): the external Km for Ca2+ ions ranged between 1 and 5 μM, while the internal Km for Ca2+ has been shown to be 0.5–1 μM. The internal and external Km values for K+ range between 1 and 5 mM, while the internal and external Km values for Na+ range from 30 to 80 mM dependent on the presence of competing alkali cations. The transport stoichiometry has been determined to be four Na+ ions in exchange for one Ca2+ and one K+ ion for NCKX1 protein in situ (Cervetto et al. 1989; Schnetkamp et al. 1989) and NCKX2 proteins expressed in cell lines (Szerencsei et al. 2001).
Functional Analysis Through Gene Deletion Experiments and Through Analysis of Mutations Found in Patients with Congenital Diseases
Structure-Function Relationships of NCKX Proteins
The SLC24A gene family encodes five K+-dependent Na+/Ca2+ exchanger proteins (NCKX1–5) that play important roles in Ca2+ signaling in many tissues. Here, we have reviewed the history of their discovery, key functional properties of NCKX proteins, and the well-established physiological role in retinal photoreceptors. Next, we have summarized what has been learned about NCKX physiology through gene deletion experiments in model organisms and through investigation of mutations found in congenital diseases. Finally, we have described our current knowledge about structure-function relationships of NCKX proteins.
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