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REVIEW

K⁺-Dependent Na⁺/Ca²⁺ Exchangers: Key Contributors to Ca²⁺ Signaling

Frank Visser and Jonathan Lytton

Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta and the Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada jlytton{at}ucalgary.ca

	Abstract

 
An elevation in cytosolic Ca²⁺ is a universal signaling mechanism that controls a vast array of physiological processes. K⁺-dependent Na⁺/Ca²⁺ exchangers are a newly identified family of Ca²⁺ efflux transporters that play important and diverse roles in cellular Ca²⁺ homeostasis.

	Introduction

Top Introduction K+-Dependent and Independent... Localization Neuronal Physiology Vision Skin Pigmentation Structure and Function Regulation Perspectives References

 
Ca²⁺ acts as a ubiquitous second messenger to control a wide variety of physiological processes by relaying information within mammalian cells (6). For example, Ca²⁺ signals trigger fertilization, control development and differentiation, coordinate cellular functions, and even play roles in cell death. The large variety of functional effects are dictated by the spatial and temporal nature of the Ca²⁺ signals and by the cellular context in which they occur, that is, the tissue and cell-specific complement of Ca²⁺ influx/efflux pathways and downstream targets determine the final outcome. Cytosolic Ca²⁺ influx typically occurs when Ca²⁺ channels localized to the plasma or endoplasmic reticular membranes open in response to various stimuli such as membrane depolarization or ligand binding (5). Ca²⁺ can be subsequently removed from the cytosol by various mechanisms: sarco/endoplasmic reticulum Ca²⁺ ATPases (SERCA) and plasma membrane Ca²⁺ ATPases (PMCA) utilize the energy from ATP hydrolysis to pump Ca²⁺ into the endoplasmic reticulum or outside the cell, respectively, and Na⁺/Ca²⁺ exchangers extrude cytosolic Ca²⁺ in exchange for extracellular Na⁺. In certain cellular contexts, such as localized signals in the dendritic spines of neurons, Ca²⁺ flux across the plasma membrane predominates over that from intracellular stores (2, 49). In such cases, PMCA proteins control the resting Ca²⁺ concentrations because of their high-affinity/low-capacity transport properties, whereas Na⁺/Ca²⁺ exchangers display low-affinity/high-capacity transport properties that are ideally suited for cytosolic clearance when Ca²⁺ is elevated following a signaling event. Detailed knowledge of the physiological and biochemical properties of calcium channels and transporters is critical to understanding the mechanisms of Ca²⁺ signaling in various cellular contexts. This review will focus on recent progress in the understanding of the physiology, function, structure, and regulation of the recently identified family of K⁺-dependent Na⁺/Ca²⁺ exchangers.

	K+-Dependent and Independent Na+/Ca2+ Exchangers

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There are two families of Na⁺/Ca²⁺ exchangers in mammalian cells: those that are K⁺-dependent (NCKX) and those that are K⁺-independent (NCX). Physiologically, NCX proteins function by extruding cytosolic Ca²⁺ in exchange for extracellular Na⁺ ions, whereas NCKX proteins extrude cytosolic Ca²⁺ and K⁺ in exchange for Na⁺ ions, which in both cases is referred to as forward mode exchange. In conditions of altered ion gradients and membrane potential, these exchangers can also mediate Ca²⁺ entry or so-called reverse mode operation. Three NCX and five NCKX isoforms have been identified by molecular cloning. NCX1 is predominantly expressed in the heart, brain, and kidney, although it is also present in a broad variety of cell types, whereas NCX2 and 3 expression is limited to brain and skeletal muscle (38, 42, 43). NCKX1 is exclusively expressed in retinal rod outer segments, whereas NCKX2 is expressed in brain and cone photoreceptors (47, 48, 59). NCKX3 and 4 display a broader pattern of tissue expression, although they are also prominently expressed in the brain (31, 36, 39). These observations indicate that NCKX2, 3, and 4 are likely to play important roles in neuronal Ca²⁺ signaling. A fifth member of the NCKX family, NCKX5, is predominantly found in intracellular compartments in skin and the pigmented epithelium of the eye, where it appears to play a critical role in pigmentation (32).

	Localization

Top Introduction K+-Dependent and Independent... Localization Neuronal Physiology Vision Skin Pigmentation Structure and Function Regulation Perspectives References

 
The first K⁺-dependent Na⁺/Ca²⁺ exchanger to be identified by molecular cloning was NCKX1 from bovine rod photoreceptors (48). Northern blotting and in situ hybridization have demonstrated that the mRNA encoding for this exchanger is found almost exclusively in rod photoreceptors (39). In contrast, when NCKX2 was cloned, the mRNA for this isoform was found in rat brain and in cone photoreceptors of chicken retina, although it was absent from most other tissues (47, 59). The localization of rat brain NCKX2 was investigated by in situ hybridization where its expression was found to be limited to neuronal cell bodies with particularly notable staining evident in all six layers of the parietal cortex, the molecular layer of the cerebellum, and throughout the neurons of the hippocampus, with CA3 neurons containing the highest levels. NCKX2 expression displayed some overlap with that of NCX1 in the neocortex and hippocampus, whereas in other regions of the brain, an almost mutually exclusive pattern of expression was observed (37, 59). The distributions of NCKX3 and 4 in mouse brain have also been investigated by in situ hybridization and were also found to be restricted to neuronal cell bodies (31, 36). NCKX3 displayed particularly strong staining throughout the thalamus, CA1 hippocampal neurons, and cortical layer IV, whereas NCKX4 expression was restricted to the anterodorsal nucleus of the thalamus, throughout the hippocampal neurons, and in cortical layers II to VI. The physiological significance of the apparently distinct localization patterns of the various NCX and NCKX isoforms in brain is not fully understood, although it could be anticipated that different neuronal subtypes will have distinct Ca²⁺-handling requirements for which the functional, thermodynamic, and regulatory properties of the various exchanger isoforms will be uniquely suited. For example, in cells where plasma membrane Ca²⁺ flux is very high and membrane potential dramatically reduced, only the NCKX exchangers would have sufficient thermodynamic driving force to extrude Ca²⁺ by virtue of their K⁺-dependence. Furthermore, the various NCX and NCKX isoforms, if present in the same cell, may be restricted to particular subcellular locations where they may play important roles in localized Ca²⁺ signaling events such as those that occur in dendritic spines (2, 49).

Interestingly, multiple tissue northern blotting and reverse-transcriptase PCR have revealed that, unlike NCKX1 and 2, NCKX3–5 display significant expression in tissues other than eye and brain (32, 39). NCKX3 displayed an almost ubiquitous pattern of expression, whereas NCKX4 expression was somewhat more limited but notable in aorta, small and large intestine, lung, and adrenal gland. These results suggest that NCKX3 and 4 may also play roles in Ca²⁺ signaling in smooth muscle cells as well as nonexcitable cell types. Recently, K⁺-dependent Na⁺/Ca²⁺ exchange activity was demonstrated in arterial smooth muscle in which NCKX3 and 4 mRNA and protein expression were also detected (10). NCKX5 also displays a broad tissue distribution, although it is most abundant in the skin and pigmented epithelium of the eye (32). Nonetheless, its presence in multiple tissues suggests that this protein may contribute to other Ca²⁺ signaling events in addition to those involved in skin pigmentation. These were the first studies to identify NCKX proteins outside the brain, but the physiological significance of these exchangers in nonneuronal tissues remains a largely unexplored area of investigation.

	Neuronal Physiology

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In neurons, Ca²⁺ signals are critical at both presynaptic and postsynaptic sites (FIGURE 1A). Presynaptically, Ca²⁺ influx occurs upon membrane depolarization via voltage-gated channels, which initiates the fusion of vesicular compartments containing neurotransmitters with the plasma membrane. Postsynaptically, Ca²⁺ entry and subsequent membrane depolarization occurs via ligand-gated channels such as the NMDA and AMPA receptors. The elevated cytosolic Ca²⁺ is subsequently removed by various plasma membrane transporters, including PMCA, NCX, and NCKX proteins (2). Several studies have investigated the relative contributions of the various Ca²⁺-clearance mechanisms in neuronal Ca²⁺ signaling and have demonstrated a prominent role for K⁺-dependent Na⁺/Ca²⁺ exchangers. In acute preparations of axon terminals from rat neurohypophysis (posterior pituitary), >60% of the Ca²⁺ clearance was mediated via Na⁺/Ca²⁺ exchangers, ~90% of which was dependent on K⁺ (34). Furthermore, a follow-up study demonstrated that NCKX2 was likely to be responsible for the observed K⁺-dependent Ca²⁺ clearance from these neurons and that the NCKX contribution was localized to the axon terminals, whereas in the somata other Ca²⁺-clearance mechanisms predominated (27). A prominent contribution by K⁺-dependent Na⁺/Ca²⁺ exchangers was also recently demonstrated at the mammalian giant presynaptic terminal, the calyx of Held (26). Lastly, consistent with the in situ hybridization studies, NCKX activity has been shown to predominate in hippocampal CA1 neurons, whereas NCX activity predominated in forebrain neurons (24, 25). The results of these studies have demonstrated that NCKX proteins play important functional roles in neuronal Ca²⁺ signaling, although the precise impact of these exchangers on neuronal function is only beginning to be elucidated. Recently, knockout mice lacking NCKX2 were shown to display deficits in motor learning and memory that could be attributed to compromised hippocampal synaptic plasticity (35). Future studies on acute brain preparations and cultured neurons from mice lacking specific NCKX isoforms will more clearly define their functional roles in Ca²⁺ signaling and the subsequent impact on neuronal function.

View larger version (76K): [in this window] [in a new window]   FIGURE 1. Physiological contexts for NCKX function in Ca2+ signaling A:Ca 2+ signaling in presynaptic and postsynaptic nerve terminals. Presynaptically, Ca2+ entry occurs via voltage-operated Ca2+ channels (VOCC) and is subsequently extruded across the plasma membrane in exchange for extracellular Na+ either alone (NCX) or together with K+ ions (NCKX). Na+ entry via voltage-gated Na+ channels (Nav) inactivates both the NCX and NCKX exchangers (dashed red lines). The plasma membrane Ca2+ ATPase (PMCA) maintains a low resting level of cytosolic Ca2+. Ca 2+ entry triggers exocytosis of neurotransmitter (glutamate)-containing vesicles, resulting in glutamate release and binding to postsynaptic NMDA or AMPA receptors. Agonist binding by NMDA and AMPA receptors induces Ca2+ entry, resulting in membrane depolarization, and the elevated Ca2+ is subsequently removed by NCX and NCKX proteins. Glutamate binding to G-protein coupled metabotropic glutamate receptors (mGluR) results in activation of phospholipase C (PLC) and the production of inositol triphosphate (IP3)and diacylglycerol (DAG). IP3 can bind to IP3 receptors on the endoplasmic reticulum (ER), resulting in the release of Ca2+ that is taken back up by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). DAG activates PKC, which enhances the activity of NCKX2 (dashed green arrow). B: Ca2+ signaling in rod photoreceptor outer segments. In the dark, cGMP channels are constitutively open, allowing Na+ and Ca2+ entry and resulting in membrane depolarization. Illumination of Rhodopsin results in activation of the G-protein transducin that activates cyclic nucleotide phosphodiesterase (PDE), which subsequently hydrolyzes cGMP. The reduction in cGMP levels results in closure of cGMP-gated channels and reduced Ca2+ entry, hyperpolarizing the membrane and allowing NCKX1 to rapidly extrude Ca2+, hyperpolarizing the membrane and initiating the photoreceptor electrical response by reducing the release of inhibitory neurotransmitters at the photoreceptor synapse. Rapid NCKX1-mediated Ca2+ extrusion results in reduced Ca2+ levels, activating Guanylyl cyclase, which resynthesizes cGMP to re-open the cGMP-gated channels. C: putative Ca2+ entry mechanism across the melanosomal membrane. Vesicles derived from the ER and trans-golgi network (TGN) undergo a four-stage process to become fully mature melanosomes, a process that apparently requires Ca2+ ions. The acidic interior of the maturing melanocyte is generated by the action of the V-ATPase proton pump, which drives Na+ uptake via a Na+/H+ exchanger (NHE), generating a Na+ gradient utilized by NCKX5 for melanosomal Ca2+ uptake.

	Vision

Top Introduction K+-Dependent and Independent... Localization Neuronal Physiology Vision Skin Pigmentation Structure and Function Regulation Perspectives References

Cone outer segments are less sensitive, display faster kinetics, and respond to a broader range of light intensities than rods. These differential characteristics can be attributed, in part, to differences in Ca²⁺ handling processes and suggest that rods and cones possess different NCKX isoforms (30). The cloning of NCKX2 from chicken retina, which is abundant in cone photoreceptors, suggested a role for this exchanger in cone physiology that was analogous to that of NCKX1 in rod photoreceptors (47). An NCKX2 paralog was also recently cloned from the retina of striped bass, and isolated cone outer segments were shown to contain K⁺-dependent Na⁺/Ca²⁺ exchange activity (45). However, NCKX2 knockout mice did not display any obvious deficits in either cone number or function, suggesting that either other Ca²⁺ efflux pathways compensated for the absence of NCKX2 or that NCKX function plays a more subtle role in Ca²⁺ homeostasis in cone photoreceptors that would require more detailed analyses to elucidate (35).

	Skin Pigmentation

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Recently the fifth member of the NCKX family, NCKX5, was identified as being mutated in zebrafish, which display the "golden" phenotype due to impaired skin pigmentation. Introduction of wild-type zebrafish or human NCKX5 constructs into these mutant fish was able to restore normal skin pigmentation (32). It was subsequently found that, in the human orthologs of NCKX5, a single nucleotide polymorphism resulting in the conversion of the highly conserved Ala 111 to Thr was strongly associated with individuals of European descent, whereas individuals of African and Asian descent contained the conserved ancestral allele. Recombinant NCKX5 is strongly expressed in skin and eye tissue and localizes to intracellular membranes, possibly melanosomes or premelanosomal compartments, which is in sharp contrast to the plasma membrane localization of the previously identified NCX and NCKX isoforms. It has been shown that Ca²⁺ uptake into maturing melanosomes is ATP-independent, insensitive to P-type ATPase inhibitors, but strongly dependent on a H⁺ gradient generated by the putative V-type H⁺-ATPase and that several subunits of this enzyme have been localized to the melanosomal membrane, as have intracellular Na⁺/H⁺ exchangers (4, 52, 56). Based on this evidence, it has been postulated that the acidic interior of maturing melanosomes drives Na⁺ uptake via Na⁺/H⁺ exchangers, which provide the driving force for Ca²⁺ uptake mediated by NCKX5 (FIGURE 1C). Although some early studies have suggested that the Ca²⁺ content of melanosomes is positively correlated with the extent of pigmentation and that Ca²⁺ is also required for melanosome motility (12, 17, 46, 51), the specific role of Ca²⁺ transport in melanosome maturation and melanin biosynthesis remains unclear. Furthermore, the functional properties of NCKX5 and the impact of the A111T mutation on these properties have not been demonstrated. Therefore, the currently proposed model and its impact on melanin biosynthesis requires additional experimental investigation.

	Structure and Function

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Early studies on isolated bovine rod outer segments were the first to functionally identify Na⁺/Ca²⁺ exchange activity that was dependent on K⁺ ions that were co-transported with Ca²⁺ with an apparent stoichiometry of 4 Na⁺:1 Ca²⁺ + 1 K⁺ (9, 54). These functional properties were subsequently confirmed for recombinant NCKX1 and 2 using either ⁴⁵Ca²⁺ uptake into insect cells or electrophysiological recordings in HEK293 cells (11, 55, 57). Furthermore, NCKX1 and 2 display similar apparent affinities for Ca²⁺ (~1–2 µM), K⁺ (15–40 mM), and Na⁺ (20–60 mM) (11, 47). Based on sequence identity, the other NCKX family members are expected to display the same stoichiometry. Recombinant NCKX3 and 4 have been demonstrated to be electrogenic (31, 36) and were recently shown to display similar apparent affinities for Ca²⁺ and Na⁺ as NCKX2 but much higher affinities for K⁺ (~1 mM) (61).

The various NCKX isoforms share the same overall predicted membrane topology, with 11 transmembrane segments (TMs 0–10), the first of which (TM 0) is thought to be cleaved by a signal peptidase after protein translation (20) (FIGURE 2). Glycosylation-scanning mutagenesis and cysteine-accessibility experiments involving NCKX2 have confirmed that, in the mature protein, both the NH₂ and COOH termini are extracellular, the NH₂-terminal extracellular loop is glycosylated, and TMs 1–5 are connected to TMs 6–10 via a large cytoplasmic loop (8, 29). There is also some evidence that NCKX2 may form a homo- or hetero-oligomer, but the functional significance of this finding is unknown (19, 65).

View larger version (96K): [in this window] [in a new window]   FIGURE 2. Predicted transmembrane topology model of NCKX2 NCKX family members are thought to share a common topology model, which has been experimentally verified for NCKX2. The first (TM 0) of 11 transmembrane segments is thought to be cleaved by a putative signal peptidase, leaving a mature protein containing two clusters of 5 TMs connected by a large cytoplasmic loop. The NH2 terminal extracellular loop is glycosylated, and the large cytoplasmic loop is alternatively spliced in NCKX2 and 4. NCKX2 contains 3 predicted PKC consensus phosphorylation sites (T166, T476, and S504) that have been attributed to phorbol ester-induced enhancement of activity. The -repeats are indicated in red and are thought to contain critical residues for cation liganding and transport, most notably E188, N215, D548, and D575. T551 of NCKX2 is an important determinant of apparent K+ affinity.

In the folded NCKX proteins, the -repeats are thought to be oppositely oriented and contain amino acid residues critical for cation liganding and translocation. The mechanism of NCKX-mediated exchange activity has not been determined in detail but is likely to follow a sequential transport mechanism. The exchanger protein likely contains a single cation binding pocket that can accommodate either, 4 Na⁺ ions or 1 Ca²⁺ + 1 K⁺ to form a "transport competent" complex that is capable of conformational change from inward-facing to outward facing or vice-versa. Moreover, NCKX proteins are "obligate exchangers", meaning that the empty carrier cannot undergo such conformational changes and requires bound substrate cations to do so.

Evidence that the same amino acid residues are involved in liganding of both K⁺ and Ca²⁺ in NCKX proteins is based on the observations that mutations of key residues in the -repeats often result in parallel decreases in affinity for both cations (21). A survey of the currently available literature from mutagenesis studies on NCX and NCKX family proteins suggests that there are four critical residues, one in the middle of each of the TMs in the -repeats that contribute to forming the cation binding pocket (FIGURE 2). In NCKX2, E188 and D548 in TMs 2 and 7, respectively, have been shown to be virtually irreplaceable and D575 in TM 8, when converted to the corresponding residue in NCX (Asn), renders NCKX2 capable of K⁺-independent Na⁺/Ca²⁺ exchange (21, 22, 62). Mutational analyses of N143 of NCX1 revealed that this residue can only tolerate substitution by Asp (44). Therefore, the corresponding NCKX2 residue (N215) may be a fourth critical residue involved in cation liganding, although its impact on NCKX function has not been reported. In NCKX2, sulfhydryl cross-linking studies have revealed that E188, D548, and D575, although far apart in the primary sequence, are close together in the folded protein where they could form the K⁺ and Ca²⁺ binding pocket (28). Thr 551 of NCKX2, which is close to D548 in the primary sequence, was recently shown to be responsible for the apparent low K⁺ affinity of NCKX2 compared with that of NCKX3 and 4 (61) (FIGURE 2).

Each of the four critical residues is preceded by a stretch of small side chain amino acid residues (i.e., Ala, Ser, Gly) that may form flexible loop regions within the otherwise helical transmembrane segments to confer enhanced freedom of movement to the critical cation binding residues. Similar structural motifs have been observed in the substrate binding pockets of the crystal structures of several transporters including the glutamate transporter, Na⁺/H⁺ exchanger, the Na⁺/Leucine co-transporter, and SERCA (18, 58, 63, 64).

Although progress in the area of structure/function of NCKX proteins has been made, there are still many questions that need to be addressed, such as the arrangement of the transmembrane helices, the identification of Na⁺-liganding residues, and the detailed functional properties of forward-mode NCKX activity. Moreover, although many studies have provided fairly compelling evidence that certain amino acid residues of NCKX proteins are critical for exchanger function, detailed structural studies clearly demonstrating direct interactions with K⁺ and/or Ca²⁺ have not been reported. Knowledge of the structural and functional properties of the various NCKX isoforms will be critical to fully understand their physiological roles.

	Regulation

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Although very little is known about regulation of the genes encoding NCKX proteins, genetic regulation of NCX1 has been thoroughly studied and is fairly well understood. The NCX1 gene is under the control of tissue-specific promoters that contain regulatory elements that respond to tissue-specific transcriptional regulation (3, 40, 41, 53). In contrast, the promoter regions of the genes encoding NCKX proteins have not been analyzed in any detail for transcription factor binding sites or tissue-specific elements (31, 60). The regions encoding the large cytosolic loop of NCX1 are alternatively spliced to yield protein products that are subject to differential functional regulation by cations (13, 14). NCKX2 and 4 were found to be alternatively spliced, with the longer forms possessing an additional 17 or 19 amino acid residues, respectively, in the large cytosolic loops, although there are no obvious functional consequences and no detailed analyses of possible differential regulation have been performed (36, 47, 59). However, NCKX2 was recently shown to become inactivated in a time- and Na⁺-dependent manner, a phenomenon that has already been well described for NCX1 (16), suggesting that NCKX2 is also subject to ionic regulation (1). Physiologically, high internal Na⁺ levels that result from persistent neuronal membrane depolarization may inactivate NCKX2, leading to persistent Ca²⁺ elevation. It is currently not known whether Na⁺-dependent inactivation is also an intrinsic property of other NCKX family members.

NCKX2, 3, and 4 all possess several consensus sites for protein kinase C (PKC) phosphorylation in their putative cytoplasmic loops, although these sites are not conserved between the isoforms. When HEK293 cells expressing NCKX2, 3, or 4 were stimulated with phorbol esters, only NCKX2 displayed increased functional activity, whereas NCKX3 and 4 were unaffected (33). It was subsequently determined that the Ca²⁺-independent PKC isoform, which predominates in neurons, was responsible for the phorbol ester-induced enhancement of activity and that mutation of the PKC consensus sites (T166, T476, and S504) to Ala abrogated this effect (FIGURE 2). It was also shown that PKC stimulation resulted in increased phosphorylation of NCKX2, which suggested that this protein may be a direct substrate for this enzyme. In neurons, activation of metabotropic glutamate receptors (mGluRs) would result in diacylglycerol and inositol triphosphate (IP₃) production and hence activation of PKC and Ca²⁺ release from internal stores via IP₃ receptors. PKC activation would presumably enhance the NCKX2 activity and hence Ca²⁺ clearance in this context, although the prevalence and importance of this potential mechanism requires further study (FIGURE 1A).

Another mode by which these exchangers might be regulated is through the formation of intermolecular complexes that may dictate their localization and/or functional activity and thus impact their influence on Ca²⁺ homeostasis. When NCKX2 is subjected to polyacrylamide gel electrophoresis under non-reducing conditions, the protein migrates as a high molecular weight species, whereas under reducing conditions it migrates at a molecular weight that is roughly consistent with that predicted from its primary sequence (19, 65). Subjecting the protein to reducing conditions was shown to stimulate its functional activity (8). One intriguing possibility is that the Cys residue responsible (C395) forms a disulfide bond with another molecule of unknown identity that may regulate its activity. Furthermore, it is possible that other NCKX isoforms may have diverse interacting partners that regulate their trafficking and localization. Clearly, these interesting questions warrant further investigation.

	Perspectives

Top Introduction K+-Dependent and Independent... Localization Neuronal Physiology Vision Skin Pigmentation Structure and Function Regulation Perspectives References

 
Since the molecular cloning of NCKX1 in 1992, there has been a molecular explosion of information regarding these interesting exchangers, resulting in the identification of five gene products with distinct tissue, cellular, and subcellular localizations. Important progress has also been made in the knowledge of their detailed functional properties and their structure/function relationships. The unique physiological roles of the exchanger isoforms are only beginning to be elucidated, but what has been discovered so far suggests that the overall impact of NCKX function has not been fully appreciated. Although preliminary, the physiological contributions of NCKX activity to Ca²⁺ signaling have been demonstrated in neurons, rod, and cone photoreceptors and implied in skin pigmentation. Many additional studies will be required to gain a more detailed understanding of the respective mechanisms involved. Therefore, the key questions for the future concern the details of the unique functional and regulatory properties of the NCKX isoforms and how these properties within their endogenous physiological contexts determine NCKX contributions to the control of various Ca²⁺-dependent cellular processes.

	References

Top Introduction K+-Dependent and Independent... Localization Neuronal Physiology Vision Skin Pigmentation Structure and Function Regulation Perspectives References

 

1. Altimimi HF, Schnetkamp PP. Na⁺-dependent inactivation of the retinal cone/brain Na⁺/Ca²⁺-K⁺ exchanger NCKX2. J Biol Chem 282: 3720–3729, 2006.[CrossRef][ISI][Medline]
2. Augustine GJ, Santamaria F, Tanaka K. Local calcium signaling in neurons. Neuron 40: 331–346, 2003.[CrossRef][ISI][Medline]
3. Barnes KV, Cheng G, Dawson MM, Menick DR. Cloning of cardiac, kidney, and brain promoters of the feline ncx1 gene. J Biol Chem 272: 11510–11517, 1997.[Abstract/Free Full Text]
4. Basrur V, Yang F, Kushimoto T, Higashimoto Y, Yasumoto K, Valencia J, Muller J, Vieira WD, Watabe H, Shabanowitz J, Hearing VJ, Hunt DF, Appella E. Proteomic analysis of early melanosomes: identification of novel melanosomal proteins. J Proteome Res 2: 69–79, 2003.[CrossRef][ISI][Medline]
5. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529, 2003.[CrossRef][ISI][Medline]
6. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21, 2000.[CrossRef][ISI][Medline]
7. Cai X, Lytton J. The cation/Ca²⁺ exchanger superfamily: phylogenetic analysis and structural implications. Mol Biol Evol 21: 1692–1703, 2004.[Abstract/Free Full Text]
8. Cai X, Zhang K, Lytton J. A novel topology and redox regulation of the rat brain K⁺-dependent Na⁺/Ca²⁺ exchanger, NCKX2. J Biol Chem 277: 48923–48930, 2002.[Abstract/Free Full Text]
9. Cervetto L, Lagnado L, Perry RJ, Robinson DW, McNaughton PA. Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients. Nature 337: 740–743, 1989.[CrossRef][Medline]
10. Dong H, Jiang Y, Triggle CR, Li X, Lytton J. Novel role for K⁺-dependent Na⁺/Ca²⁺ exchangers in regulation of cytoplasmic free Ca²⁺ and contractility in arterial smooth muscle. Am J Physiol Heart Circ Physiol 291: H1226–H1235, 2006.[Abstract/Free Full Text]
11. Dong H, Light PE, French RJ, Lytton J. Electrophysiological characterization and ionic stoichiometry of the rat brain K⁺-dependent NA⁺/CA²⁺ exchanger, NCKX2. J Biol Chem 276: 25919–25928, 2001.[Abstract/Free Full Text]
12. Drager UC. Calcium binding in pigmented and albino eyes. Proc Natl Acad Sci USA 82: 6716–6720, 1985.[Abstract/Free Full Text]
13. Dunn J, Elias CL, Le HD, Omelchenko A, Hryshko LV, Lytton J. The molecular determinants of ionic regulatory differences between brain and kidney Na⁺/Ca²⁺ exchanger (NCX1) isoforms. J Biol Chem 277: 33957–33962, 2002.[Abstract/Free Full Text]
14. Dyck C, Omelchenko A, Elias CL, Quednau BD, Philipson KD, Hnatowich M, Hryshko LV. Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na⁺-Ca²⁺ exchanger. J Gen Physiol 114: 701–711, 1999.[Abstract/Free Full Text]
15. Fain GL, Matthews HR, Cornwall MC, Koutalos Y. Adaptation in vertebrate photoreceptors. Physiol Rev 81: 117–151, 2001.[Abstract/Free Full Text]
16. Hilgemann DW, Matsuoka S, Nagel GA, Collins A. Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation. J Gen Physiol 100: 905–932, 1992.[Abstract/Free Full Text]
17. Howard J. Calcium enables photoreceptor pigment migration in a mutant fly. J Exp Biol 113: 471–475, 1984.[Free Full Text]
18. Hunte C, Screpanti E, Venturi M, Rimon A, Padan E, Michel H. Structure of a Na⁺/H⁺ antiporter and insights into mechanism of action and regulation by pH. Nature 435: 1197–1202, 2005.[CrossRef][Medline]
19. Kang K, Bauer PJ, Kinjo TG, Szerencsei RT, Bonigk W, Winkfein RJ, Schnetkamp PP. Assembly of retinal rod or cone Na⁺/Ca²⁺-K⁺ exchanger oligomers with cGMP-gated channel subunits as probed with heterologously expressed cDNAs. Biochemistry 42: 4593–4600, 2003.[CrossRef][Medline]
20. Kang K, Schnetkamp PP. Signal sequence cleavage and plasma membrane targeting of the retinal rod NCKX1 and cone NCKX2 Na⁺/Ca²⁺-K⁺ exchangers. Biochemistry 42: 9438–9445, 2003.[CrossRef][Medline]
21. Kang KJ, Kinjo TG, Szerencsei RT, Schnetkamp PP. Residues contributing to the Ca²⁺ and K⁺ binding pocket of the NCKX2 Na⁺/Ca²⁺-K⁺ exchanger. J Biol Chem 280: 6823–6833, 2005.[Abstract/Free Full Text]
22. Kang KJ, Shibukawa Y, Szerencsei RT, Schnetkamp PP. Substitution of a single residue, Asp575, renders the NCKX2 K⁺-dependent Na⁺/Ca²⁺ exchanger independent of K⁺. J Biol Chem 280: 6834–6839, 2005.[Abstract/Free Full Text]
23. Kaupp UB, Seifert R. Cyclic nucleotide-gated ion channels. Physiol Rev 82: 769–824, 2002.[Abstract/Free Full Text]
24. Kiedrowski L. High activity of K⁺-dependent plasmalemmal Na⁺/Ca²⁺ exchangers in hippocampal CA1 neurons. Neuroreport 15: 2113–2116, 2004.[CrossRef][ISI][Medline]
25. Kiedrowski L, Czyz A, Baranauskas G, Li XF, Lytton J. Differential contribution of plasmalemmal Na/Ca exchange isoforms to sodium-dependent calcium influx and NMDA excitotoxicity in depolarized neurons. J Neurochem 90: 117–128, 2004.[CrossRef][ISI][Medline]
26. Kim MH, Korogod N, Schneggenburger R, Ho WK, Lee SH. Interplay between Na⁺/Ca²⁺ exchangers and mitochondria in Ca²⁺ clearance at the calyx of Held. J Neurosci 25: 6057–6065, 2005.[Abstract/Free Full Text]
27. Kim MH, Lee SH, Park KH, Ho WK, Lee SH. Distribution of K ⁺-dependent Na⁺/Ca²⁺ exchangers in the rat supraoptic magnocellular neuron is polarized to axon terminals. J Neurosci 23: 11673–11680, 2003.[Abstract/Free Full Text]
28. Kinjo TG, Kang K, Szerencsei RT, Winkfein RJ, Schnetkamp PP. Site-directed disulfide mapping of residues contributing to the Ca²⁺ and K⁺ binding pocket of the NCKX2 Na⁺/Ca²⁺-K⁺ exchanger. Biochemistry 44: 7787–7795, 2005.[CrossRef][Medline]
29. Kinjo TG, Szerencsei RT, Winkfein RJ, Kang K, Schnetkamp PP. Topology of the retinal cone NCKX2 Na/Ca-K exchanger. Biochemistry 42: 2485–2491, 2003.[CrossRef][Medline]
30. Korenbrot JI, Rebrik TI. Tuning outer segment Ca²⁺ homeostasis to phototransduction in rods and cones. Adv Exp Med Biol 514: 179–203, 2002.[ISI][Medline]
31. Kraev A, Quednau BD, Leach S, Li XF, Dong H, Winkfein R, Perizzolo M, Cai X, Yang R, Philipson KD, Lytton J. Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3. J Biol Chem 276: 23161–23172, 2001.[Abstract/Free Full Text]
32. Lamason RL, Mohideen MA, Mest JR, Wong AC, Norton HL, Aros MC, Jurynec MJ, Mao X, Humphreville VR, Humbert JE, Sinha S, Moore JL, Jagadeeswaran P, Zhao W, Ning G, Makalowska I, McKeigue PM, O’Donnell D, Kittles R, Parra EJ, Mangini NJ, Grunwald DJ, Shriver MD, Canfield VA, Cheng KC. SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310: 1782–1786, 2005.[Abstract/Free Full Text]
33. Lee JY, Visser F, Lee JS, Lee KH, Soh JW, Ho WK, Lytton J, Lee SH. Protein kinase C-dependent enhancement of activity of rat brain NCKX2 heterologously expressed in HEK293 cells. J Biol Chem 281: 39205–39216, 2006.[Abstract/Free Full Text]
34. Lee SH, Kim MH, Park KH, Earm YE, Ho WK. K⁺-dependent Na⁺/Ca²⁺ exchange is a major Ca²⁺ clearance mechanism in axon terminals of rat neurohypophysis. J Neurosci 22: 6891–6899, 2002.[Abstract/Free Full Text]
35. Li XF, Kiedrowski L, Tremblay F, Fernandez FR, Perizzolo M, Winkfein RJ, Turner RW, Bains JS, Rancourt DE, Lytton J. Importance of K⁺-dependent Na⁺/Ca²⁺-exchanger 2, NCKX2, in motor learning and memory. J Biol Chem 281: 6273–6282, 2006.[Abstract/Free Full Text]
36. Li XF, Kraev AS, Lytton J. Molecular cloning of a fourth member of the potassium-dependent sodium-calcium exchanger gene family, NCKX4. J Biol Chem 277: 48410–48417, 2002.[Abstract/Free Full Text]
37. Li XF, Lytton J. Differential expression of Na/Ca exchanger and Na/Ca + K exchanger transcripts in rat brain. Ann NY Acad Sci 976: 64–66, 2002.[Free Full Text]
38. Li Z, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP, Lifton RP, Philipson KD. Cloning of the NCX2 isoform of the plasma membrane Na ⁺-Ca²⁺ exchanger. J Biol Chem 269: 17434–17439, 1994.[Abstract/Free Full Text]
39. Lytton J, Li XF, Dong H, Kraev A. K⁺-dependent Na⁺/Ca²⁺ exchangers in the brain. Ann NY Acad Sci 976: 382–393, 2002.[Abstract/Free Full Text]
40. Menick DR, Xu L, Kappler C, Jiang W, Withers P, Shepherd N, Conway SJ, Muller JG. Pathways regulating Na⁺/Ca²⁺ exchanger expression in the heart. Ann NY Acad Sci 976: 237–247, 2002.[Abstract/Free Full Text]
41. Nicholas SB, Yang W, Lee SL, Zhu H, Philipson KD, Lytton J. Alternative promoters and cardiac muscle cell-specific expression of the Na⁺/Ca²⁺ exchanger gene. Am J Physiol Heart Circ Physiol 274: H217–H232, 1998.[Abstract/Free Full Text]
42. Nicoll DA, Longoni S, Philipson KD. Molecular cloning and functional expression of the cardiac sarcolemmal Na⁺-Ca²⁺ exchanger. Science 250: 562–565, 1990.[Abstract/Free Full Text]
43. Nicoll DA, Quednau BD, Qui Z, Xia YR, Lusis AJ, Philipson KD. Cloning of a third mammalian Na ⁺-Ca²⁺ exchanger, NCX3. J Biol Chem 271: 24914–24921, 1996.[Abstract/Free Full Text]
44. Ottolia M, Nicoll DA, Philipson KD. Mutational analysis of the alpha-1 repeat of the cardiac Na⁺-Ca²⁺ exchanger. J Biol Chem 280: 1061–1069, 2005.[Abstract/Free Full Text]
45. Paillart C, Winkfein RJ, Schnetkamp PP, Korenbrot JI. Functional characterization and molecular cloning of the K⁺-dependent Na⁺/Ca²⁺ exchanger in intact retinal cone photoreceptors. J Gen Physiol 129: 1–16, 2007.[Abstract/Free Full Text]
46. Panessa BJ, Zadunaisky JA. Pigment granules: a calcium reservoir in the vertebrate eye. Exp Eye Res 32: 593–604, 1981.[CrossRef][ISI][Medline]
47. Prinsen CF, Szerencsei RT, Schnetkamp PP. Molecular cloning and functional expression of the potassium-dependent sodium-calcium exchanger from human and chicken retinal cone photoreceptors. J Neurosci 20: 1424–1434, 2000.[Abstract/Free Full Text]
48. Reilander H, Achilles A, Friedel U, Maul G, Lottspeich F, Cook NJ. Primary structure and functional expression of the Na/Ca, K-exchanger from bovine rod photoreceptors. EMBO J 11: 1689–1695, 1992.[ISI][Medline]
49. Sabatini BL, Oertner TG, Svoboda K. The life cycle of Ca ²⁺ ions in dendritic spines. Neuron 33: 439–452, 2002.[CrossRef][ISI][Medline]
50. Saier MH Jr, Eng BH, Fard S, Garg J, Haggerty DA, Hutchinson WJ, Jack DL, Lai EC, Liu HJ, Nusinew DP, Omar AM, Pao SS, Paulsen IT, Quan JA, Sliwinski M, Tseng TT, Wachi S, Young GB. Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim Biophys Acta 1422: 1–56, 1999.[Medline]
51. Salceda R, Riesgo-Escovar JR. Characterization of calcium uptake in chick retinal pigment epithelium. Pigment Cell Res 3: 141–145, 1990.[CrossRef][ISI][Medline]
52. Salceda R, Sanchez-Chavez G. Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium. Cell Calcium 27: 223–229, 2000.[CrossRef][ISI][Medline]
53. Scheller T, Kraev A, Skinner S, Carafoli E. Cloning of the multipartite promoter of the sodium-calcium exchanger gene NCX1 and characterization of its activity in vascular smooth muscle cells. J Biol Chem 273: 7643–7649, 1998.[Abstract/Free Full Text]
54. Schnetkamp PP, Basu DK, Szerencsei RT. Na⁺-Ca²⁺ exchange in bovine rod outer segments requires and transports K⁺. Am J Physiol Cell Physiol 257: C153–C157, 1989.[Abstract/Free Full Text]
55. Sheng JZ, Prinsen CF, Clark RB, Giles WR, Schnetkamp PP. Na⁺-Ca²⁺-K⁺ currents measured in insect cells transfected with the retinal cone or rod Na⁺-Ca²⁺-K⁺ exchanger cDNA. Biophys J 79: 1945–1953, 2000.[ISI][Medline]
56. Smith DR, Spaulding DT, Glenn HM, Fuller BB. The relationship between Na⁺/H⁺ exchanger expression and tyrosinase activity in human melanocytes. Exp Cell Res 298: 521–534, 2004.[CrossRef][ISI][Medline]
57. Szerencsei RT, Prinsen CF, Schnetkamp PP. Stoichiometry of the retinal cone Na/Ca-K exchanger heterologously expressed in insect cells: comparison with the bovine heart Na/Ca exchanger. Biochemistry 40: 6009–6015, 2001.[CrossRef][Medline]
58. Toyoshima C, Nakasako M, Nomura H, Ogawa H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405: 647–655, 2000.[CrossRef][Medline]
59. Tsoi M, Rhee KH, Bungard D, Li XF, Lee SL, Auer RN, Lytton J. Molecular cloning of a novel potassium-dependent sodium-calcium exchanger from rat brain. J Biol Chem 273: 4155–4162, 1998.[Abstract/Free Full Text]
60. Tucker JE, Winkfein RJ, Murthy SK, Friedman JS, Walter MA, Demetrick DJ, Schnetkamp PP. Chromosomal localization and genomic organization of the human retinal rod Na-Ca+K exchanger. Hum Genet 103: 411–414, 1998.[CrossRef][ISI][Medline]
61. Visser F, Valsecchi V, Annunziato L, Lytton J. Exchangers NCKX2, NCKX3, and NCKX4: identification of Thr-551 as a key residue in defining the apparent K⁺ affinity of NCKX2. J Biol Chem 282: 4453–4462, 2007.[Abstract/Free Full Text]
62. Winkfein RJ, Szerencsei RT, Kinjo TG, Kang K, Perizzolo M, Eisner L, Schnetkamp PP. Scanning mutagenesis of the alpha repeats and of the trans-membrane acidic residues of the human retinal cone Na/Ca-K exchanger. Biochemistry 42: 543–552, 2003.[CrossRef][Medline]
63. Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E. Crystal structure of a bacterial homologue of Na⁺/Cl^–-dependent neurotransmitter transporters. Nature 437: 215–223, 2005.[CrossRef][Medline]
64. Yernool D, Boudker O, Jin Y, Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431: 811–818, 2004.[CrossRef][Medline]
65. Yoo SS, Leach S, Lytton J. Studies on the oligomeric state of the sodium/calcium + potassium exchanger NCKX2. Ann NY Acad Sci 976: 94–96, 2002.[Free Full Text]

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