Molecular Aspects of Medicine 34 (2013) 455–464

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Molecular Aspects of Medicine

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Review

The SLC24 gene family of Na+/Ca2+–K+ exchangers: From sight and smellto memory consolidation and skin pigmentation q

Paul P.M. SchnetkampDepartment of Physiology & Pharmacology, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary,Canada AB T2N 4N1

Guest Editor Matthias A. HedigerTransporters in health and disease (SLC series)

a r t i c l e i n f o

Article history:Received 3 January 2012Accepted 9 March 2012

Keywords:SLC24NCKXNa+/Ca2+–K+ exchangerPhotoreceptorsOlfactionCalcium homeostasisSkin pigmentation

0098-2997/$ - see front matter � 2012 Elsevier Ltdhttp://dx.doi.org/10.1016/j.mam.2012.07.008

q Publication in part sponsored by the Swiss NatUniversity of Bern, Switzerland; Director Matthias A

E-mail address: pschnetk@ucalgary.ca

a b s t r a c t

Members of the SLC24 gene family encode K+-dependent Na+/Ca2+ exchangers (NCKX) thatutilize both the inward Na+ and outward K+ gradients to extrude Ca2+ from cells. There arefive human SLC24 genes that play a role in biological process as diverse as vision in retinalrod and cone photoreceptors, olfaction, skin pigmentation and at least three of the fivegenes are also widely expressed in the brain. Here I review the functional, physiologicaland structural features of NCKX proteins that have emerged in the past few years.

� 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4562. NCKX family members, tissue distribution and splice variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4563. Physiology of NCKX proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4564. Functional characteristics of NCKX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4585. Evidence supporting the alternating access model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4586. Km values for Ca2+-, K+- and Na+-dependent exchange fluxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4587. Topological model and residues important for cation transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4598. Crystal structure of a bacterial Na/Ca exchanger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4609. Genetics: link to disease, association and gene deletion studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46110. Future perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

. All rights reserved.

ional Science Foundation through the National Center of Competence in Research (NCCR) TransCure,. Hediger; Web: http://www.transcure.ch.

456 P.P.M. Schnetkamp / Molecular Aspects of Medicine 34 (2013) 455–464

1. Introduction

Calcium ions are the most commonly used intracellular messengers in biology and precise temporal and spatial control oftransient increases in cytosolic free [Ca2+] is critical to this function. A rise in cytosolic free [Ca2+] may occur by Ca2+ entrythrough either plasma membrane Ca2+ channels (e.g. voltage-gated channels, cyclic nucleotide-gated channels, TRP or P2Xchannels, store-operated channels or neurotransmitter-gated channels) or through Ca2+ release from intracellular stores viathe IP3 or ryanodine receptors. In all cases Ca2+ diffuses down its electrochemical gradient to increase cytosolic free [Ca2+].Restoration of cytosolic free [Ca2+] to resting levels of 50–100 nM requires uphill Ca2+ transport coupled to a source of en-ergy. The latter is provided either by 1) the hydrolysis of ATP in members of the plasma membrane (PMCA) Ca2+ pumps andCa2+ pumps located in the sarco/endoplasmic reticulum (SERCA); or 2) by coupling to the large Na+ gradient that existsacross the plasma membrane of most cells in the case of the Na+/Ca2+ exchangers.

Na+/Ca2+ exchange was first described in the nineteen sixties and is thought to be the predominant mechanism for Ca2+

extrusion in tissues that require fast Ca2+ signaling such as most if not all excitable tissues. For a historical overview on Na+/Ca2+ exchange see Blaustein and Lederer (1999). Na+/Ca2+ exchangers come in two flavours, those that only use the inwardNa+ gradient to extrude Ca2+ (NCX, members of the SLC8 gene family)(Quednau et al., 2004) and those that use both the in-ward Na+ gradient and the outward K+ gradient to extrude Ca2+ (NCKX, members of the SLC24 gene family). NCKX was firstdescribed in the outer segments of retinal rod photoreceptors in the late nineteen eighties and was reviewed in my 2004contribution to the SLC transporter overview (Schnetkamp, 2004). The objective of this review is to provide an update onour current knowledge on NCKX, focusing in particular on new genetic studies on SLC24 genes and on new insights intothe residues that are important for NCKX function. For more detailed reviews on the physiology and function of NCKX pro-teins see Altimimi and Schnetkamp (2007a) and Lytton (2007). SLC24 (and SLC8) belong to the CaCA family of Ca2+-cationantiporters which are part of the CPA superfamily (http://www.tcdb.org/superfamily.php).

2. NCKX family members, tissue distribution and splice variants

Table 1 summarizes several features of the SLC24 gene family encoding the NCKX proteins. Splice variants of several SLC24genes have been reported, e.g. several splice variants have been reported for a rat ortholog of SLC24A1 (Poon et al., 2000) andseveral of the various mammalian SLC24A1 cDNAs that were cloned would appear to represent different splice variants (Coo-per et al., 1999), for example the human NCKX1 sequence we reported lacks exon III encoding 18 residues (Tucker et al.,1998). A transport stoichiometry of 4Na+/1Ca2++1K+ has been determined experimentally for NCKX1 and NCKX2 (see below),and, based on sequence identity in areas important for transport (see below), it is reasonable to suggest that the same trans-port stoichiometry will be observed for NCXK3-5. On the other hand, NCKX6 is now known as NCLX and a member of the CCX(Ca2+/cation exchanger) branch of the CaCA (Ca2+/cation antiporter) superfamily (Lytton, 2007). More recently, NCLX hasbeen shown to be an essential component of mitochondrial Na+/Ca2+ exchange which is thought to operate as an electrogenicNa+/Ca2+ or Li+/Ca2+ exchanger, not known to either require or transport K+ (Palty et al., 2010). NCLX is now classified as amember of the SLC8 gene family (SLC8B1) (Khananshvili, 2013).

3. Physiology of NCKX proteins

Apart from a number of recent genetic studies discussed later very few studies have directly assessed the in situ role ofNCKX proteins in tissues and reported direct measurements of NCKX-mediated cation fluxes except for the well-documentedstudies on the role of NCKX1 in the outer segments of retinal rod photoreceptors (ROS) (reviewed in Schnetkamp (1995a)and Lagnado et al. (1992)). The transport stoichiometry was determined to be 4Na+:1 Ca2+ + 1K+ for NCKX1 in ROS (Schnetk-amp et al., 1989; Cervetto et al., 1989) as well for NCKX2 expressed in cell lines (Szerencsei et al., 2001; Dong et al., 2001).This means that Ca2+ extrusion from cells is driven by both inward Na+ and outward K+ gradients, and, in view of the elec-trogenic nature of transport, also by the negative inside membrane potential. It also means that Na+/Ca2+–K+ exchangers canmaintain larger Ca2+ gradients when compared with the NCX Na+/Ca2+ exchangers operating at a 3:1 stoichiometry; more-over, Na+/Ca2+–K+ exchangers will not as readily reverse from the Ca2+ efflux mode to Ca2+ influx mode upon strong mem-brane depolarization and/or reduction of the transmembrane Na+ gradient when compared with the Na+/Ca2+ exchanger.This would make NCKX proteins a preferred choice for cells that show long-lived depolarizations due to sustained inwardNa+ currents which are likely to decrease the transmembrane Na+ gradient as well, thereby reducing two of the three com-ponents of the driving force to extrude Ca2+ from cells via NC(K)X exchangers. This scenario certainly applies to retinal rodand cone photoreceptors which both operate by modulating a sustained inward Na+ and Ca2+ current carried by cGMP-gatedchannels (Fain et al., 2001). The cGMP-gated channels and the NCKX1 Na+/Ca2+–K+ exchanger are the only two ion transport-ers present in the plasma membrane of the outer segments from retinal rod photoreceptors making this an ideal preparationfor their study (Schnetkamp et al., 1991b). In bovine ROS, NCKX1 has been shown to be present as a dimer (Schwarzer et al.,1997) that is part of a large multi-protein complex with the heteromultimeric cGMP-gated channel (Bauer and Drechsler,1992) and two small proteins located in the rims of the internal disk membranes (Poetsch et al., 2001). When expressedin insect high five cells or HEK293 cells, both NCKX1 and NCKX2 were shown to form homo-dimers and bind to the CNGAsubunit of both the rod and cone cGMP-gated channels, respectively (Kang et al., 2003). In darkness, a steady inward current

Table 1SLC24–Na+/(Ca2+–K+) exchanger family. For detailed information about the SLC gene tables, please visit: http://www.bioparadigms.org.

Humangene name

Proteinname

Aliases Predominantsubstrates

Transport type/coupling ions⁄)

Tissue distribution and cellular/subcellularexpression

Link to disease Human genelocus

Sequenceaccession ID

SLC24A1 NCKX1 RODX, HsT17412, KIAA0702 Na+, Ca2+, K+ E/4Na+/Ca2+–K+ Rod photoreceptors, platelets/plasma membrane Congenital stationarynight blindness

15q22 NM_004727

SLC24A2 NCKX2 Na+, Ca2+, K+ E/4Na+/Ca2+–K+ Brain, retinal cone photoreceptors, retinalganglion cells/plasma membrane

9p22-p13 NM_020344

SLC24A3 NCKX3 Na+, Ca2+, K+ E/4Na+/Ca2+–K+ Brain, aorta, uterus, intestine/plasma membrane 20p13 NM_020689SLC24A4 NCKX4 SHEP6, FLJ38852 Na+, Ca2+, K+ E/4Na+/Ca2+–K+ Brain, olfactory neurons, aorta, lung, thymus/

plasma membrane14q32.12 NM_153646

NM_153647NM_153648

SLC24A5 NCKX5 JSX, SHEP4 Na+, Ca2+, K+ E/4Na+/Ca2+–K+ Retinal pigment epithelium, brain, thymus, skin/intracellular

15q21.1 NM_205850

References:Original version of the SLC table:Schnetkamp PP. The SLC24 Na(+)/Ca(2+)-K(+) exchanger family: vision and beyond. Pflugers Arch. 2004 Feb;447(5):683–8.

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carried by Na+ (80–90%) and Ca2+ ions (10–20%) results in a sustained membrane depolarization and in a long lasting in-crease in cytosolic free [Ca2+] mediated by the balance between Ca2+ influx via cGMP-gated channels and Ca2+ extrusionvia the NCKX1 Na+/Ca2+–K+ exchanger. Absorption of light by rhodopsin initiates an enzymatic cascade that results in thehydrolysis of cGMP, closure of cGMP-gated channels, membrane hyperpolarization and a lowering of cytosolic free [Ca2+]by the continued operation of NCKX1 in the absence of any further Ca2+ influx. Lowering of cytosolic free [Ca2+] activatesthe enzyme responsible for cGMP synthesis and this negative feedback loop is thought to mediate part of the process of lightadaptation. For a more detailed review of the role of cGMP-gated channels, Na+/Ca2+–K+ exchangers and cytosolic free [Ca2+]on rod and cone physiology consult Fain et al. (2001) and references therein.

SLC24A2 transcripts have been found in the cone photoreceptors of chicken (Prinsen et al., 2000), human (Prinsen et al.,2002), and striped bass retinas (Paillart et al., 2007) and the only direct measurements of Na+/Ca2+–K+ exchange in cone pho-toreceptors so far were reported in the latter study. Based on these results it is thought that NCKX2 plays the same role incone photoreceptors as that played by NCKX1 in rod photoreceptors. NCKX2 transcripts are also found throughout the brain(Tsoi et al., 1998) and K+-dependent Na+/Ca2+ exchange as well as K+-independent Na+/Ca2+ exchange have been reported tocoexist in cultured neurons (Czyz and Kiedrowski, 2002; Kiedrowski, 2004; Kim et al., 2005; Lee et al., 2002). However,SLC24A3 and SLC24A4 transcripts are also found throughout the brain (Kraev et al., 2001; Li et al., 2002) and it is not clearwhich NCKX isoform is responsible for the reported K+-dependent Na+/Ca2+ exchange. This is corroborated by the observa-tion that neurons cultured from Slc24a2�/� mice retained both K+-independent Na+/Ca2+ exchange (�70% of total Na+/Ca2+

exchange) and as well as K+-dependent Na+/Ca2+ exchange (reduced by 40–50% in the Slc24a2�/� animals), most likelyreflecting the presence of NCKX3 and/or NCKX4 (Li et al., 2006).

4. Functional characteristics of NCKX

Like most solute carriers, NCKX transporters are thought to operate by the alternating access mechanism. The alternatingaccess mechanism proposes that a single set of ion/substrate binding sites are alternately exposed to the extracellular andintracellular space, respectively. In the case of obligatory exchangers like NCKX, Na+ ions bind to binding sites exposed to theextracellular environment and this binding is mandatory to induce a conformational transition after which the bound Na+

ions become inaccessible to the extracellular environment and accessible to the intracellular environment for release intothe cytosol. The Na+/Ca2+–K+ exchanger displays an absolute selectivity for Na+ in this step of the exchange transport processas no other cation can replace Na+. After release of Na+ into the intracellular space, the empty binding sites are now availableto bind Ca2+ and K+ from the cytosol, and, again, this binding is obligatory to induce the next conformational change afterwhich the bound Ca2+ and K+ are available for release into to the extracellular space.

The alternating access mechanism of NCKX-mediated transport has two functional consequences of great physiologicalsignificance. Firstly, transport is bidirectional and depends on the prevailing cation gradients, i.e. upon reversal or collapseof the transmembrane Na+ gradient, NCKX can act as a Ca2+ (and K+) importer and may mediate a large Ca2+ influx into cells;in addition to Ca2+ extrusion via forward exchange, Kþout- and Naþin-dependent Ca2+ influx or reverse exchange has been widelyobserved both in situ (Schnetkamp, 1986; Schnetkamp et al., 1989; Cervetto et al.,1989) and in cells transfected with cDNA’sencoding various NCKX proteins (Sheng et al., 2000; Dong et al., 2001; Altimimi and Schnetkamp, 2007b). As a matter of fact,Kþout-dependent Ca2+ influx via reverse exchange has become the signature for evidence of NCKX activity and is by far themost widely used NCKX assay. In terms of pathophysiology, conditions that compromise the metabolic integrity of cells likeischemic episodes may cause a (partial) collapse of the transmembrane Na+ gradient and a concomitant runaway Ca2+ influxvia reverse exchange most likely resulting in cell death in cells which express NCKX (and/or NCX) proteins as a predominantmechanism for Ca2+ extrusion. In a similar vein, experimental manipulations of extracellular Na+ and Ca2+ concentrations incells or tissues that express NCKX are expected to result in rapid changes in cytosolic free [Ca2+].

5. Evidence supporting the alternating access model

In addition to the forward and reverse exchange modes discussed above, the alternating access model would also predict theoccurrence of self-exchange fluxes, i.e. Ca2+ + K+:Ca2+ + K+ and Na+:Na+, and these fluxes have been observed in ROS using radio-active 45Ca, 86Rb (a K substitute) and 22Na fluxes with rates comparable to either reverse or forward Na+/Ca2+–K+ exchange(Schnetkamp et al., 1991b). Furthermore, the alternating access model would predict the presence of a single set of bindingsites that accommodates either four Na+ ions or one Ca2+ ion and one K+ ion. This implies that Ca2+ and K+ should compete withNa+ at least for some of these common binding site(s) as was examined in detail for NCKX1 in retinal ROS preparations. Based ona so-called Dixon analysis Ca2+ was shown to compete with two Na+ ions for a common binding site, whereas other alkali cat-ions such as Li+ and K+ did not compete with Ca2+ (Schnetkamp et al., 1991a). In contrast, Li+ and K+ did compete with Na+ for acommon site suggesting this/these would be the site(s) binding the ‘‘other two’’ Na+ ions (Schnetkamp and Szerencsei, 1991).

6. Km values for Ca2+-, K+- and Na+-dependent exchange fluxes

As a result from the above described competitive interactions between various cations for binding to the NCKX cationbinding sites, the Km values observed for the Na+, Ca2+ and K+ dependencies of Na+/Ca2+–K+ exchange fluxes are expected

P.P.M. Schnetkamp / Molecular Aspects of Medicine 34 (2013) 455–464 459

to depend greatly on the exact ionic composition of the medium in which such values were obtained. For example, the Km forNa+ is expected to be higher when measured in a medium that contains either Ca2+ or Li+ or K+ compared with media thatcontain sucrose or salts of organic cations such as choline or tetramethylammonium as the major osmotic component of themedium. Conversely, the Km for Ca2+ is expected to be higher when measured in a medium that contains Na+ but is not ex-pected to be affected by the presence of Li+ or K+ (although the presence of K+ is required for transport, K+ does not competewith Ca2+). Consistent with the above expectations, reported Km values for Na+ range from values of 30–40 mM in the ab-sence of any competing cations to values of 60–80 mM in the presence of high external Li+ or K+ to values of >100 mM inthe presence of physiological extracellular Ca2+; Hill coefficients ranged typically between 2 and 3. Km values reported forboth extracellular and intracellular Ca2+ in the absence of competing cations range from 1 to 3 lM. It should be pointedout that a rather steep pH dependence has also been reported for the external Km for Ca2+ (Schnetkamp, 1995b) providingfor yet another complexity. Given the range of experimental methods and conditions used it would appear that the Km valuesobserved for both Ca2+ and Na+ were very similar for in situ measurements in ROS and for a range of different NCKX clonesexpressed in cell lines. The reported Km values for K+ are more variable. A significant K+-independent Na+/Ca2+ exchange hasbeen observed for in situ NCKX1 in ROS (Schnetkamp et al., 1991a,b) and for NCKX2 expressed in cell lines (Prinsen et al.,2000) when the external medium is free of alkali cations. Hence, the standard assay adopted for K+-dependent reverseNa+/Ca2+ exchange uses a medium containing 150 mM LiCl and this resulted in a �fivefold increase in the K+ Km of NCKX2expressed in cell lines (Prinsen et al., 2000). In a high LiCl medium, the reported external Km values for K+ range between 2and 10 mM either when measured for in situ NCKX1 or for various NCKX clones expressed in cell lines with NCKX2 as anoticeable exception. Values reported for NCKX2 show a considerably greater range with some studies reporting valuesof �5 mM (e.g. (Kang et al., 2005a,b; Paillart et al., 2007) but others reporting a value of >30 mM, e.g.(Lee et al., 2002;Visseret al., 2007). The reason for this discrepancy is unclear.

7. Topological model and residues important for cation transport

Hydropathy analysis reveals that NCKX1-5 all share a very similar structural pattern consisting of two largehydrophilic loops and twelve predominantly hydrophobic segments long enough to form a putative alpha helical trans-membrane segment (TMS). The TMS are arranged in clusters of 1, 5 and 6 TMS, respectively, with the two large hydro-philic loops separating these clusters. A very short hydrophilic segment precedes putative TMS 1 at the N-terminus andfollowing putative TMS 12 another very short hydrophilic segment completes the NCKX2 protein at the C-terminus. Fur-ther bioinformatics analysis suggests that the first TMS is either a cleavable signal peptide or an uncleaved signal anchor.Expression of NCKX2 in cell lines invariably results in two populations of NCKX2 protein, one representing full-lengthprotein and the other NCKX2 protein from which the signal peptide has been cleaved; furthermore, deletion of the signalpeptide prevented correct trafficking to the plasma membrane (Kang and Schnetkamp, 2003). Further studies led to thetopological model illustrated in Fig. 1: in addition to the signal peptide at the N-terminus NCKX2 is thought to containtwo clusters of five TMS each and one of the putative TMS is thought to be a hydrophobic segment located in the cytosol,perhaps at the membrane surface as illustrated (Kinjo et al., 2003). These two clusters of 5 TMS each contains all thesequence elements conserved among the five NCKX genes. The two central TMS of each cluster are thought to have arisenfrom an ancient gene duplication event and are oriented in an inverted fashion. These so called a1 and a2 repeats are themost conserved parts of the NCKX sequence and are also the only parts of the NCKX sequence that show some, albeit verylimited, sequence similarity to members of the SLC8 gene family of K+-independent Na+/Ca2+ exchangers. Scanning muta-genesis of the a repeats revealed the presence of twenty-five residues which, when substituted, caused a >80% reductionin transport activity compared to WT NCKX2 while protein expression and plasma membrane trafficking were not af-fected (Winkfein et al., 2003); all these residues are conserved between human NCKX1 though 5. This large number ofcritical residues is likely a reflection that NCKX needs to accommodate binding of four Na+ ions, and, not surprisingly,most of these twenty-five residues are acidic residues (Asp, Glu) or hydrophilic residues (Ser, Thr, Asn) whose side chainscan provide coordinating sites for Ca2+ and Na+. Other residues among this group are glycine and proline residues whichmay provide for flexible joints and helix-helix contact sites (Gly) or induce helix-loop-helix motifs often found in ionbinding sites (Pro). Consistent with their role in cation binding, substitution of most of the above residues also causedsignificant shifts in the Km for Ca2+, Na+ and K+ (Kang et al., 2005a; Altimimi et al., 2010). This provides further supportfor the alternating access model which postulates the presence of a single set of cation binding sites that can accommo-date either four Na+ or one Ca2+ plus one K+. Aspartate 575 proved to be a single residue critical for K+ transport as theAsp575Asn and Asp575Cys substitutions resulted in mutant NCKX2 proteins that completely lost their requirement for K+

and displayed K+-independent Na+/Ca2+ exchange (Kang et al., 2005a). In addition to Asp575 critical for K+ transport, weproposed that Glu188 and Asp548 are the two residues most critical for Ca2+ binding and transport (Kang et al., 2005b).Fig. 1 illustrates many of the residues critical for NCKX function superimposed on our topological model of NCKX2. Thesekey residues are located in in the two alpha repeats far apart in the linear NCKX sequence. Using site-directed disulfidemapping we showed that Glu188 and Ser185 are in close proximity to Asp548, Ser552 and Asp575 consistent with thenotion that the two alpha repeats are in close proximity and that these residues are part of the cation binding pocket ofNCKX2 (Kinjo et al., 2005).

Fig. 1. Current topological model of NCKX proteins. The NCKX protein embedded in a phospholipid bilayer membrane is depicted. Hydrophobic segments of�20 residues that are thought to span the membrane in the form of a helices (TMS) are shown as bars whereas hydrophilic stretches of protein are shown assolid lines. The TMS at the N-terminus is a signal peptide that can be cleaved by a signal peptidase (SPase). The bars labeled 1–5 and 6–10 are thought to besets of five TMS each, while the bar labeled ‘‘hydrophobic segment’’ is thought to be located near the cytosolic surface but not spanning the membrane. Theblue-colored segments represent the so-called alpha repeats discussed in the text. Highlighted are also some of the residues shown to be important forcation binding and cation transport as discussed in the text: red, acidic residues; orange, hydrophilic residues; blue, glycine and proline residues.

460 P.P.M. Schnetkamp / Molecular Aspects of Medicine 34 (2013) 455–464

8. Crystal structure of a bacterial Na/Ca exchanger

Very recently, the first crystal structure of a member of CaCA gene family was reported (Liao et al., 2012). NCX_Mj fromMethanococcus jannaschii was shown to be a Na/Ca exchanger after purification and reconstitution, and the overall topologyof this bacterial Na/Ca exchanger is identical to our topological model of NCKX2 although sequence identity is limited to only25 out of the 75 residues that make up the alpha 1 and alpha 2 repeats. More significantly, 14 of those 25 residues were

Fig. 2. Overall structure of the bacterial Na/Ca exchanger NCX_Mj. Transmembrane helices 1 through 5 are colored pale orange while helices 6 through 10are colored aquamarine. Alpha repeat residues conserved between mammalian NCKX2 and NCX_Mj are highlighted in red, while the acidic residues Glu54,Glu213 and Asp240 are highlighted in blue. The latter three residues are homologous to critical NCKX2 residues Glu188, Asp548 and Asp575 as discussed inthe text. The green sphere represents the bound Ca2+. Left panel: view from the plane of the membrane. Right panel: looking down on the membrane fromthe cytosolic side.

Retina

Skin pigmentation

Uterus

Hypertension

NCKX1

NCKX2

NCKX3

NCKX4

SLC24Gene Family

Northern European

Hair colour

Iris colour

Smell

Artery

Hippocampus

TGN

PigmentEpithelium

Discs

RodPhotoreceptors

ConePhotoreceptors

Melanosomes

?

NCKX5

Fig. 3. Physiological settings for human and mouse NCKX proteins.

P.P.M. Schnetkamp / Molecular Aspects of Medicine 34 (2013) 455–464 461

recently shown by our laboratory to be important for Na+ binding to NCKX2 (Altimimi et al., 2010). Moreover, the residueshomologous to Glu188 and Asp548 were shown to contribute four of the six oxygen ligands that make up the Ca2+ bindingsite of NCX_Mj, consistent with their role in Ca2+ binding to NCKX2 (see above). Fig. 2 illustrates the NCX_Mj structure.

9. Genetics: link to disease, association and gene deletion studies

Fig. 3 illustrates the various physiological settings in which NCKX proteins have been shown or suggested to play impor-tant roles.

SLC24A1 Inherited retinal disease in humans is highly variable and mutations in more than 200 different genes, themajority encoding proteins in rod photoreceptors, have been associated with various forms of retinal blinding disease(http://www.sph.uth.tmc.edu/RetNet/). In an extensive screen of 800 patients with hereditary retinal disease, not associatedwith any previously known gene, 27 novel sequence variants were found in the SLC24A1 gene and six were considered to bepossibly pathogenic (Sharon et al., 2002). In all six cases this concerned only a single patient and a single allele and patho-genicity could not be proven. More recently, members of a Pakistani family were shown to have a two base pair deletion inexon2 that results in a frame shift and is predicted to result in premature termination of the NCKX1 protein (Riazuddin et al.,2010). This homozygous deletion segregated with the condition of congenital stationary night blindness (CSNB) in an auto-

462 P.P.M. Schnetkamp / Molecular Aspects of Medicine 34 (2013) 455–464

somal recessive manner while heterozygous or unaffected family members were free of visual symptoms. Unlike manyinherited retinal diseases, CNSB is a nonprogressive disorder that results in impaired night vision but no progressive degen-eration of the retina with age is observed.

SLC24A2 No studies have appeared yet that link sequence variants of the SLC24A2 gene to any disease or altered humantraits/phenotype. (Sharon et al., 2002) reported 14 novel sequence changes in the SLC24A2 gene of patients with a variety ofretinal cone dystrophies but none were considered to be pathogenic. Deletion of the nckx2 gene in mice resulted in signif-icant deficits in motor learning and memory and changes in hippocampal synaptic plasticity as judged by a significant loss oflong term potentiation in CA1 pyramidal neurons (Li et al., 2006). Induction of ischemic brain damage through artery occlu-sion in WT mice with nckx2�/�mice suggested that NCKX2 could provide protection against ischemic brain damage (Cuomoet al., 2008).

SLC24A3 Very little is known about the physiology of NCKX3. For the uterus it has been reported that expression ofNCKX3 transcript and protein is regulated by steroid hormones during the menstrual cycle (Yang et al., 2011) while NCKX3transcript and protein levels in the kidney were found to be higher for female mice compared with males (Lee et al., 2009). Ina recent association study on proteins important for salt-sensitive hypertension the involvement of the NCX1 and NCKX3plasma membrane Na+/Ca2+–(K+) exchangers was assessed (Citterio et al., 2011). SNP’s in both the SLC8A1 and SLC24A3 geneswere associated with pathophysiological variations in systolic blood pressure.

SLC24A4 Two studies have reported that upstream SNP’s in the SLC24A4 gene are associated with hair and eye color inEuropean populations suggesting that NCKX4 may play a role in pigmentation in hair follicle and uveal melanocytes (Sulemet al., 2007; Pospiech et al., 2011). Slc24a4�/�mice were recently reported to have a deficit in olfactory neurons that affectsthe duration and adaptation of the olfactory response (Stephan et al., 2012). Signal transduction in olfactory sensory neurons(OSN) is mediated by an increase in cAMP levels which leads to an increase in cytosolic free [Ca2+] which in turn activatesCa2+-activated Cl� channels. The latter carry the majority of the current that causes OSN membrane depolarization. It hasbeen well established that Na+/Ca2+ exchange is the predominant mechanism of Ca2+ extrusion in OSN although it was re-ported to be of the K+-independent variety (Reisert et al., 2007;Antolin and Matthews, 2007). In these studies Na+/Ca2+ ex-change was measured indirectly taking advantage of its ability to change cytosolic free [Ca2+] as measured by concomitantchanges in Ca2+-activated Cl� current. However, the Slc24a4 knockout study clearly shows that nearly all of the Na+/Ca2+ ex-change controlling OSN response termination through closure of Ca2+-activated Cl� channels could be accounted for byNCKX4 as it is eliminated in the Slc24a4�/� mice. As Ca2+ is responsible for olfactory adaptation, the Slc24a4�/� miceshowed a severely reduced ability to located buried food pellets and also had a reduced body weight (Stephan et al., 2012).

SLC24A5 The ortholog of SLC24A5 in the zebrafish has been shown to be the gene responsible for the so called ‘‘golden’’phenotype. These zebrafish display a severely reduced pigmentation both in the skin and in the retinal pigment epitheliumdue to a large reduction in the number and pigmentation of melanophores (Lamason et al., 2005). Pigmentation could berestored by the human SLC24A5 gene. When a tagged version of the human SLC24A5 cDNA was expressed in the pigmentedhuman MNT1melanoma cell line this resulted in intracellular localization of NCKX5 protein and no plasma membraneexpression was observed (Lamason et al., 2005). This is in contrast with the NCKX1-4 proteins which are thought to functionas plasma membrane proteins and, when overexpressed in cell lines, can be functionally detected in the plasma membrane,although a large amount of internally localized NCKX protein is seen as well.

SLC24A5 transcripts are also abundant in pigmented cells of humans. The WT SLC24A5 gene was found in peoples of Afri-can, indigenous North American and East Asian descent whereas a nonsynonymous SNP on both alleles of the SLC24A5 genewas found to be fixed in people from European descent and lighter skin pigmentation in admixed populations correlated withthe SNP (Lamason et al., 2005; Stokowski et al., 2007). Deletion of the Slc24a5 gene in mice did not result in any changes of coatcolor but did result in a marked reduction in pigmentation of the retinal pigment epithelium (Vogel et al., 2008). Thus, NCKX5is a critical participant in pigmentation as found in the retinal pigment epithelium and epidermal melanocytes. Two furtherstudies confirmed the absence of NCKX5 protein and activity in the plasma membrane of primary melanocytes and insteadlocated the NCKX5 protein either to late-stage melanosomes (Chi et al., 2006) or the trans Golgi network (Ginger et al., 2008).

10. Future perspective

Apart from the well-understood role of NCKX1 in retinal rod photoreceptors much of the physiological role of the otherfamily members remains to be elucidated. Recent genetic studies described above have revealed a tantalizingly wide rangeof physiological roles for NCKX proteins from memory consolidation to olfaction and skin pigmentation. A key missing toolfor future studies on NCKX physiology is the current lack of compounds that selectively inhibit NCKX, never mind selectivelyinhibit a specific NCKX isoform.

Acknowledgement

Research on the SLC24 gene family in my laboratory is funded through operating grant MOP-81327 from the CanadianInstitutes for Health Research. My thanks are due to Dr. Lorraine Aha for her careful reading of the text.

P.P.M. Schnetkamp / Molecular Aspects of Medicine 34 (2013) 455–464 463

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