Potassium-dependent sodium-calcium exchanger (NCKX) isoforms and neuronal function

Cell Calcium 86 (2020) 102135

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Potassium-dependent sodium-calcium exchanger (NCKX) isoforms and neuronal function

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Mohamed Tarek Hassan, Jonathan Lytton* Department of Biochemistry & Molecular Biology, Libin Cardiovascular Institute and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: K-dependent Na/Ca-exchangers Neuronal function

K+-dependent Na+/Ca2+-exchangers (NCKX) are a relatively recently described five-member gene family of transporters which play a quantitatively significant role in neuronal Ca2+ transport. In this review we highlight the important individual contributions these transporters make to cellular Ca2+ homeostasis and neuronal function. Notably, different members of the family make distinct, non-redundant, contributions to critical behavioural pathways. In particular, NCKX proteins regulate the kinetics, termination and adaptation of Ca2+ signals in sensory transduction neurons in the olfactory and visual systems. Similar contributions to shaping the spatial and temporal features of Ca2+ signals in neurons at other key brain locations have important consequences for the circuitry influencing control of satiety, for experience-dependent motor learning and spatial working memory retention, as well as in the protection of neurons in the face of toxic stimuli. NCKX proteins are also key contributors to a variety of events in other tissues. The connection between NCKX isoform function and human phenotype and disease is an emerging area, and we anticipate that future research will reveal rich new details in the coming years.

1. Calcium homeostasis and the molecular toolkit Calcium ion (Ca2+) is a well-known universal second messenger that plays a particularly critical role in all aspects of neuronal cell physiology and pathology. It has been established that Ca2+ signals are either directly responsible for, or acutely regulate, events such as: developmental morphogenesis and axonal projection; neural pruning and apoptosis; neurotransmitter release; synaptic plasticity; membrane excitability; gene regulation; adaptation to sensory signals; and more. Specificity among these many pathways is thought to be achieved in cells by the nature of the sensors expressed in combination with precise and complex spatial and temporal control over the Ca2+ signals themselves. In part, this is accomplished through the distinct molecular “toolkit” of Ca2+ handling proteins that each cell expresses [1,2]. Fig. 1 illustrates many of these proteins, and is a useful visual reference for the following few paragraphs. Ca2+ signals can be conceptually divided into initiation; buffering and sensing; termination; and restitution phases, which follow oneanother chronologically [1,3]. In the initiation phase, a signal at the plasma membrane – typically a change in voltage, agonist binding to its receptor, activation via a second messenger, or physical stretch – induces the opening of one or more Ca2+-permeable channels, resulting in influx of Ca2+ into the cytosol. In addition to influx across the plasma ⁎

membrane, signals initiated here can be transmitted to and result in release of Ca2+ from the endoplasmic or sarcoplasmic reticulum stores. The resulting increase in intracellular Ca2+ is both buffered and sensed by a number of Ca2+-binding proteins, most notably those containing EF-hand or C2-domain binding motifs, which ultimately convey the Ca2+ signal into a downstream response. Signal termination involves both the cessation of Ca2+ influx and release, the movement of Ca2+ into various cellular sinks, such as mitochondria, and the return of Ca2+ to its sources through the action of various pumps and exchangers. Finally, restitution helps reset the cell back into its basal state, as Ca2+ is released more slowly from buffers and sinks, stores are replenished, and receptors released from inactive states. While the shape and kinetics of the Ca2+ signal are influenced by all of the above factors and molecules, of particular interest here are the pathways that terminate the signal and remove Ca2+ from the cytosol. Two classes of transporters are responsible, pumps and exchangers. Pumps, which include the plasma membrane Ca2+-ATPase, PMCA [4], the endoplasmic or sarcoplasmic reticulum Ca2+-ATPase, SERCA, and the secretory pathway Ca2+-ATPase, SPCA [5], are relatively low capacity transporters that are tuned to controlling Ca2+ in the 100 nM range, and thus are principally responsible for maintaining Ca2+ under resting or restitution conditions. Na+/Ca2+-exchangers on the other hand, which couple the uphill movement of Ca2+ to the cellular ion

Corresponding author at: Health Research Innovation Centrem, 3280 Hospital Drive NW, Calgary, AB, T2N 3Z8, Canada. E-mail address: [email protected] (J. Lytton).

https://doi.org/10.1016/j.ceca.2019.102135 Received 31 October 2019; Received in revised form 25 November 2019; Accepted 26 November 2019 Available online 09 December 2019 0143-4160/ © 2019 Elsevier Ltd. All rights reserved.

Cell Calcium 86 (2020) 102135

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Fig. 1. K+-dependent Na+/Ca2+-exchangers (NCKX) are key mediators of cellular Ca2+ homeostasis. Cellular Ca2+ signals are initiated by the entry of Ca2+ across the plasma membrane or the release from intracellular stores through various channels. The Ca2+ signal is then terminated by uptake into mitochondria, sequestration in the endoplasmic reticulum (ER) or efflux across the plasma membrane. NCKX proteins are major contributors to Ca2+ efflux across the plasma membrane, and may also be important for Ca2+ transport across some intracellular membranes, such as the Golgi complex in the case of NCKX5. VOC, ROC, SOC: voltage-, receptor- and second messenger-operated channels. IP3R and RyR: inositol-trisphosphate- and Ryanodinereceptor channels of the ER. SERCA and PMCA: sarcoplasmic or endoplasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPase. NCX, NCLX and NCKX: Na+/Ca2+-exchanger, Na+/Ca2++Li+-exchanger of the mitochondria, Na+/Ca2++K+-exchanger. Mito and MCU: mitochondria and mitochondrial Ca2+ uniporter.

mining experiments identified the five members of this family [16–20]. Expression of NCKX1 (SLC24A1) is restricted largely to the rod photoreceptors; NCKX2 (SLC24A2) is selectively expressed in neurons (including cone photoreceptors); NCKX3 (SLC24A3) and NCKX4 (SLC24A4) are also present in the brain, but their expression is not restricted to neurons, and they are also broadly expressed in other tissues [21]. NCKX5 (SLC24A5) has some selective expression in the brain, but its main physiological function is thought to be related to melanosome maturation in pigmented epithelial cells, rather than neurons [22]. The existence of robust Na+/Ca2+-exchange activity in neurons was established more than 50 years ago [6]. However, the realization that a substantial fraction of this activity is encoded by NCKX, as opposed to NCX, exchangers was only appreciated relatively recently after the genes and their products had been identified [13]. Consequently, the study of NCKX proteins and their physiological functions in neurons has lagged behind the studies of other Ca2+ transporters. Although their relative contribution to Ca2+ flux varies from region to region in the brain, as much as 40–60 % of total Ca2+ flux is mediated by NCKX proteins [7,23–27]. NCKX proteins, with their high turnover rate and relatively low affinity, are particularly well suited for extruding Ca2+ during and shortly after the peak in its concentration that occurs with

gradients established by the Na+.K+-ATPase, have a very high capacity, but somewhat lower affinity, and become the principal means of Ca2+ extrusion when cytosolic levels rise significantly during a signal [6,7].

2. The Calcium-cation antiporter superfamily and K+-dependent Na+/Ca2+-exchangers Ca2+ transporting exchangers comprise a large superfamily of Ca2+/cation antiporter (CaCA) genes which are conserved among all domains of life [8,9]. A schematic phylogenetic tree of the for CaCA members is shown in Fig. 2. Three of the five clades of the CaCA superfamily are expressed in mammals: CCX, NCX, and NCKX [10]. The CCX (SLC8B) branch, which includes NCLX [11], is thought to encode the mitochondrial Na+/Ca2+-exchanger, that operates with a 3 Na+ to 1 Ca2+ ion stoichiometry. The NCX (SLC8A) branch codes for three Na+/Ca2+-exchanger genes which share a transport stoichiometry of 3 Na+ to 1 Ca2+ ion [12]. NCKX (SLC24), the K+-dependent Na+/Ca2+exchanger, is encoded by five genes that transport 4 Na+ in exchange for 1 Ca2+ and 1 K+ ion [13]. The absolute requirement of NCKXs for K+ for transport function was first described in the outer segments of rod photoreceptors [14,15]. Subsequently, gene cloning and database 2

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Fig. 2. The Ca2+/cation antiporter superfamily and NCKX phylogeny. A phylogenetic tree of the Ca2+/cation antiporter (CaCA) superfamily illustrates the five major branches: YRBG (archaeal and prokaryotic members); CAX (containing mostly fungal and plant members); NCX (eukaryotic members including the mammalian SLC8 family); NCKX (eukaryotic members including the mammalian SLC24 family); CCX (eukaryotic members including the mammalian SLC8B family of mitochondrial exchangers). Representative members of each branch are labeled. EcoYRBG: the yrbG protein of Escherichia coli, a proton/Ca2+-exchanger. CerVCX: the yeast vacuolar proton/Ca2+-exchanger of Saccharomyces cerevisiae. AthCAX1 and 2: the plant proton/Ca2+-exchanger gene 1 and 2 products from Arabidopsis thaliana. NCX1, 2 and 3: SLC8 family members from Homo sapiens. NCKX1, 2, 3, 4 and 5: SLC24 family members from Homo sapiens. NCLX: the mitochondrial Na+/Ca2+(/Li+)-exchanger from Homo sapiens. Figure modified from Cai & Lytton (2004) [8].

that takes place in the rod and cone photoreceptor cells in the retina. Rods respond to low intensity light with a broad spectral composition, and are thus suited to vision under condition of dim light, especially at night. Cones, on the other hand, response to intense light of (in humans) three distinct spectral characteristics described as red, green and blue, and are responsible for colour vision in bright light. In both type of cells, different members of the NCKX family play an essential role in maintaining Ca2+ homeostasis that is essential in proper vision. In the dark, photoreceptors are continuously in a depolarized state due to the influx of Na+ and Ca2+ through the cGMP-gated (CNG) channels. Parallel extrusion of Ca2+ to maintain a steady state low intracellular concentration under these conditions requires an exchanger with NCKX, rather than NCX, stoichiometry. Light triggers a signaling cascade that leads to the closure of the CNG channels and inhibition of the inward depolarizing current. The consequential hyperpolarization is translated into an electric signal and the visual information is conveyed through retinal bipolar and ganglion cells, and then via the optic nerve, to the brain. Meanwhile, NCKX continues to remove Ca2+ from the cell, and the consequent drop in concentration is essential to trigger a negative feedback mechanism to reduce the signal strength through an increase in cGMP levels, and to reopen the CNG

an action potential. At this time, when the membrane potential is relatively depolarized and the Na+ gradient is reduced, the altered ionic dependence and stoichiometry of NCKX compared to NCX provides a distinct advantage. NCX, which couples 3 Na+ ions to 1 Ca2+ ion, reverses transport direction under physiological conditions during an action potential, thus contributing to Ca2+ entry, rather than exit, during this interval. NCKX, on the other hand, reverses only under quite extreme and rarely experienced shifts in membrane potential and ion gradients, and is thus poised to continue extruding Ca2+ over a broader range of conditions [21]. 3. Phototransduction in the visual system As mentioned above, NCKXs were first described, and are still best understood, in the context of vision [28]. Consequently, we will begin with a separate discussion on NCKXs in neurons of the visual system before going on to their roles in neurons in the brain. The first member of the NCKX family discovered, NCKX1, was originally isolated from bovine retina, where it is the only known mechanism of Ca2+ extrusion from the outer segments of the rod photoreceptors. Visual perception requires the conversion of a light signal into an electric one, a process 3

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channels. The modulation of Ca2+ levels thus helps control the response, kinetics and adaptation of the photoreceptor cells, and is key to enable them to function during continuous illumination over a broad range of light intensity [29,30]. Consistent with expectations for its critical role in phototransduction in rods, a mutation in NCKX1 (SLC24A1) was found to result in recessive congenital stationary night blindness (CSNB), a condition associated with defective night (dim light) vision [31]. Perhaps somewhat surprisingly – given that defective Ca2+ homeostasis might be expected to result in cell death – CSNB is a non-progressive and nondegenerative disease. An Nckx1 knockout mouse model for CSNB has also been developed [32]. Rods from these mice demonstrated a large decrease in the signal amplitude, slow recovery after a light signal, and impaired adaptation. The reduced adaptation and slow signal recovery are anticipated from a defect in Ca2+ extrusion in the Nckx1-/- mice. Adaptive changes in other pathways appear to blunt the anticipated effect of Nckx1 knockout on rod Ca2+ homeostasis, potentially providing an explanation for the non-degenerative nature of CSNB. The phototransduction mechanism in cone photoreceptors is similar to that in rods, although the two photoreceptor types differ significantly in their sensitivity and response kinetics. These physiological differences likely result from the distinct anatomical features and different isoforms of all the proteins – opsins, G-proteins, channels, exchangers, etc – involved in light response expressed in cones compared to rods [33]. Two exchanger isoforms, NCKX2 and NCKX4, are present in the cones in contrast to only NCKX1 in the rods. The fast kinetics and the wider dynamic range of Ca2+ in cones, in addition to the fact that Ca2+ constitutes a bigger portion of the inward current here compared to rods [34], may explain the need for a more efficient Ca2+ clearance system in cones that consists of two NCKX isoforms compared to one in the rods. Interestingly, despite being highly expressed in the cone photoreceptors, data from gene-targeted mice lacking NCKX2 demonstrated relatively normal cone function – especially under steady state illumination – although the kinetics of inactivation, responsiveness and adaptation were all altered [35,36]. These data suggested that another exchanger, now known to be NCKX4, also contributes to Ca2+ extrusion and photo-response modulation in cones [37]. Similar to Nckx2-/- mice, the Nckx4-/- animals displayed relatively normal cone responsiveness, although the kinetics of response and adaptation were altered. Double Nckx2/Nckx4 knockout animals had a dramatically reduced photo-response and, with age, their cones were susceptible to morphological change and cell death in a manner that paralleled the effect of NCKX1 loss on rod morphology and function in Nckx1-/- mice. These studies reinforce the complementary requirement for NCKX2 and NCKX4 in normal cone function and photo-responsiveness. This may help explain why no mutations in either gene have yet been linked to retinal disease in humans. It is noteworthy that both NCKX2 and NCKX4 also appear to be expressed in bipolar and ganglion cells of the retina, yet their specific physiological roles here remain unknown [37,38].

this information. 5. NCKX2: a neuronal K+-dependent Na+/Ca2+-exchanger Transcripts from the Nckx2 gene are broadly and specifically expressed in neurons throughout the brain, with particularly high abundance in hippocampal CA3 pyramidal cells, stellate cells of the cerebellar molecular layer, neurons in the thalamic medial geniculate, and deeper layers of the cerebral cortex [17,21]. Analysis of the NCKX2 protein revealed expression that was regionally variable but was typically found preferentially in neuronal processes well away from the soma, with an enrichment in dendritic structures in the hippocampus, but also in axonal structures in other brain regions [40]. Interestingly, there is a relatively high fraction of NCKX2 found within dendritic shafts, presumably associated with vesicles undergoing trafficking. In this regard, it appears that the polarized distribution of NCKX2 is regulated by endocytic removal from the dendrite surface membrane and KIF21A-mediated transport to axonal sites [41,42]. The first gene knockout in the NCKX family was for Nckx2 [35]. Ca2+ flux experiments in cultured cortical neurons from the Nckx2-/animals displayed a reduction of about 45 % in K+-dependent Na+/ Ca2+-exchange activity [35]. Furthermore, NCKX currents measured in cortical neurons are reduced by ∼30-50 % in Nckx2-/- animals [43]. Thus, while NCKX2 is a major contributor to neuronal Ca2+ homeostasis, other NCKX members, likely NCKX3 and NCKX4, must also participate. Despite this significant change in neuronal Ca2+ transport capacity, Nckx2-/- animals were healthy, fertile, and did not behave or respond differently from their wild type counterparts in any obvious manner. There was no evident pathology observed in histological examination of brain sections from these animals, nor any significant differences in neuronal number of morphology in the regions where NCKX2 was most abundant. Careful tests for learning and memory, including the Morris watermaze and the rotorod tests, revealed significant deficits in the ability of Nckx2-/- animals to improve their performance with time. Thus, NCKX2 function appears to be important for normal experiencedependent motor learning, spatial working memory, and memory retention [35,44]. The development of spatial memory and learning is thought to be initiated by the potentiation of synapses in the hippocampus. Examination of long term potentiation (LTP) and long term depression (LTD) at the CA3-CA1 Schaffer collateral synapses was examined, and a pronounced shift from LTP to LTD was observed in the Nckx2-/- mice, consistent with the behavioural observations. The difference in plasticity appeared to have a post-synaptic origin and, given the preferential expression of NCKX2 in dendritic shafts in the hippocampal stratum radiatum [40], it has been suggested that the normal role for NCKX2 at these sites is to modulate dendritic Ca2+ levels during post-synaptic stimulation, hence influencing the efficiency of EPSP summation, and thus signal transduction. A change in calmodulindependent kinase II and IV expression and activity may be responsible for such changes in synaptic responsiveness and plasticity in the Nckx2-/- mice [44]. These studies reinforce the important role that NCKX2 plays in neuronal Ca2+ homeostasis, and how a deficit, even partial, in this critical process can lead to significant neuronal dysfunction and behavioural changes. Stroke, or its experimental counterpart ischemia, is a critical pathological condition in which dys-regulation of Ca2+ homeostasis contributes to cell death and brain damage. As anticipated from the data supporting an important quantitative role of NCKX2 in neuronal Ca2+ extrusion, knockdown or knockout of Nckx2 expression results in exacerbated brain damage following focal ischemia [43]. Presumably, the protective effect of NCKX2 in the face of ischemia and oxygen deprivation (which induce an intracellular increase in Ca2+, an increase in Na+, and a decrease in K+ levels), is because the 4 Na+ to (1 Ca2+ and 1 K+) stoichiometry of this transporter ensures there is still sufficient thermodynamic driving force to allow continued Ca2+ extrusion

4. K+-dependent Na+/Ca2+-exchangers in the brain A role for K+-dependent Na+/Ca2+-exchange activity in neuronal Ca homeostasis had been suggested almost 30 years ago [39]. However, it was not until the molecular cloning of the NCKX members, NCKX2, NCKX3 and NCKX4, and the discovery that all were abundantly expressed in brain tissue, that there was a resurgent interest in uncovering the distinct physiological function(s) affiliated with these isoforms. As noted above, data suggest that NCKXs constitute the dominant mechanism of neuronal Ca2+ clearance, particularly in processes away from the soma [26,27,40]. However, because there are no good pharmacological tools to investigate this directly, definitive experiments awaited the arrival of gene knockout models. The following sections describe our current knowledge for the role of each NCKX protein in neuronal function. Fig. 3 is a schematic which summarizes 2+

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Fig. 3. Neuronal functions of K+-dependent Na+/Ca2+ exchangers. K+-dependent Na+/Ca2+-exchangers (NCKX) important for neuronal Ca2+ homeostasis are linked to distinct neuron functions. In the retina, NCKX1 in rods, and NCKX2 and NCKX4 in cones, regulate the duration and adaptation of the visual response. Mutations in NCKX1 in humans are linked to congenital stationary night blindness (CSNB). NCKX4 plays a similar modulatory role in the olfactory sensory neurons, and mice lacking the protein show deficits in olfactory response. Nckx4-/mice also demonstrated increased satiety and weight loss associated with constitutive activation of the melonocortin-4 receptor neurons in the paraventricular nucleus of the hypothalamus. Furthermore, NCKX4 is expressed in the apical membrane of mature ameloblasts and is essential for proper enamel mineralization. Mutations in NCKX4 in humans are associated with amelogenesis imperfecta (AI). In the hippocampus, NCKX2 is present on the dendrites of the CA1 neurons where it is responsible for integration of post-synaptic potentials. Loss of NCKX2 in mice results in defects in motor learning and spatial working memory. See text for details and citations.

thereby influencing the excitability of neighbouring neurons. Although there is not a one-to-one correspondence between different Nckx paralogs in Drosophila and their orthologs in mammalian systems, the NCKX-zydeco protein is most similar to mammalian NCKX3. Indeed, there is some evidence for NCKX3 expression in mammalian glial cells [51]. These exciting observations deserve to be following up by more detailed and careful examination of the role of NCKX3 in neuronal function/dys-function in mammalian systems. Further studies on NCKX3 in brain are eagerly anticipated.

under these conditions.

6. NCKX3: a broadly expressed K+-dependent Na+/Ca2+exchanger Robust expression of Nckx3 has been observed in brain, with particular abundance seen in hippocampal pyramidal CA1 neurons and distinct thalamic nuclei, as well as in other brain regions [18,21,45]. Nckx3 is also known to be expressed relatively broadly in other tissues, and so its expression in brain may not be restricted only to neurons. Nevertheless, at this time there are no studies which examine the role of Nckx3 in neuronal function or behavior. Recently, a Nckx3 gene knockout model was developed in mice [46] but brain function was not assessed. Instead, studies on NCKX3 function have focused on other tissues where the gene is known to be expressed, such as renal epithelial cells, and uterine endometrium [47,48]. Interestingly, the Nckx3-/mice demonstrated a modest reduction in bone density and mineral content, and an increase in circulating parathyroid hormone [46]. However, the location and mechanisms leading to these changes have not yet been revealed. Given the level of Nckx3 expression in brain, a quantitative effect on neuronal Ca2+ homeostasis would be anticipated. Moreover, the focal nature of Nckx3 expression that is very high in a restricted number of brain regions, suggests there might be important pathology or behavior changes induced by ablation of the gene, that cannot be compensated by changes in the expression of other Ca2+ transporters. Recently an NCKX homolog from Drosophila melanogaster, identified as the gene responsible for the seizure-sensitizing mutation called “zydeco”, has been described as playing a critical role in cortical glial cell Ca2+ dynamics underlying a glial-neuronal communication pathway influencing excitability [49,50]. In Drosophila the NCKX-zydeco protein appears to be exclusively expressed in glial cells. Here, NCKX-zydeco is essential for maintaining Ca2+ oscillations near the membrane that are proposed to control endocytosis and K+ buffering,

7. NCKX4: vision, olfaction and more Nckx4 was first cloned from mouse brain, where transcripts are particularly abundant and selectively enriched in pyramidal neurons of the hippocampal dentate gyrus, in glomerula and granule neurons of the olfactory bulb, in Purkinje neurons of the cerebellum, and in selected other brain regions [19]. Nckx4 expression is also strong in a variety of other tissues, particularly lung, aorta and intestine. These studies have largely been performed at the gene and transcript level because, until very recently, antibody and other tools to examine NCKX4 protein location and function were lacking or non-specific. Consequently, there is very little information regarding the NCKX4 protein itself. The development of more specific reagents to address these outstanding issues is eagerly anticipated. A physiological function for NCKX4 was first demonstrated in olfactory neurons, where targeted gene loss results in changes in signal termination and adaptation, and subsequently defects in olfactory sensation [52]. Although the details of molecular mechanism are different, the conceptual parallels between the role of NCKX4 in olfactory and that of NCKX1, 2 and 4 in visual, sensation are striking. In addition to olfactory deficits, Nckx4-/- mice also have lower body weight than their wild type counterparts, largely because they eat less [53]. Although olfactory issues may contribute to the reduction in eating and the lean phenotype, particularly in younger animals, there is clearly a 5

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The apparent key role for NCKX proteins in adaptation of signaling pathways and in neuronal plasticity suggests their activity might also be subject to allosteric regulatory processes, much like the many wellstudied events that impact function of the cardiac NCX1 protein [12]. Purinergic signaling operating via both ionotropic and metabotropic pathways is a prevalent means for modulating neuronal function in response to both neurotransmitter release and glial-initiated signals [2]. In this regard, NCKX4 activity has been shown to increase 2- to 4-fold in both a recombinant system [66] and an endogenous setting [unpublished data] when stimulated with ATP. This action is mediated via P2Y receptors, and appears to require both calmodulin-dependent kinase and protein kinase C (PKC) activity. Earlier studies had established that NCKX2, in contrast, could be stimulated via PKC activity alone, though the connection to purinergic signaling is indirect here [67]. In both instances, although direct phosphorylation of the exchanger protein was associated with stimulation of activity, phosphorylation alone was insufficient to account for the increases observed. Thus, the molecular mechanism underlying regulation of NCKX function is not yet known. Interestingly, in an unrelated series of experiments, src-family kinase activity was shown to modulate NCKX2 trafficking and cell surface residence [42]. Possibly, similar changes in protein distribution underlie other modes of regulation. In photoreceptors, NCKX activity is known to be functionally coupled to the activity of CNG channels. Various in vitro studies have documented physical associations between NCKX1 and CNGA1 and between NCKX2 and CNGA3, as well as NCKX homo-dimeric interactions, that all involve disulfide linkages [68], and which may indirectly influence exchange activity [69]. The large central cytoplasmic loops of the NCKX proteins also contain motifs for various phosphorylation events and calmodulin binding, though these are mostly not conserved between isoforms, and none are currently linked to known physiological pathways. Finally, NCKX activity has been reported to be inhibited by high concentrations of intracellular Na+ in a slow time-dependent fashion [70], though the physiological significance of this regulation is currently unclear.

primary deficit in satiety circuits of the brain that results in constitutive activation of melanocortin-4 receptor expressing neurons in the hypothalamic paraventricular nucleus that cannot be explained as a secondary response to reduced food intake [53]. A distinct role for NCKX4 in normal neuronal satiety signaling has important implications for understanding molecular mechanism in a highly clinically relevant area. This exciting and intriguing observation has only been documented in global knockout mice, and a cellular and molecular explanation for the phenotype awaits further experiments. NCKX4 has also been linked to defects in dental enamel formation, as a consequence of its function in Ca2+ secretion from the apical surface of ameloblasts [54–56]. Interestingly, abundant Nckx4 expression in other tissues – such as lung, aorta and intestine [19] – suggests an important contribution of NCKX4 to Ca2+ homeostasis in these tissues. Nckx4 is also expressed in both lens tissue [57] and melanocyte cell lines [58], and has been implicated in hair and eye pigmentation (see below). However, there are no data yet for the physiological consequence of NCKX4 expression at these sites – making these all potential areas of attention for future exploration. 8. NCKX5: the odd one out Nckx5 has been considered something of an outlier to the NCKX family since the discovery that mutations in this gene lead to the “golden” skin pigmentation mutation in zebrafish, and that polymorphisms underlie a fraction of variation in human skin colouration [20]. Unlike other NCKX family members, NCKX5 appears not to be expressed on the cell surface, but rather in intracellular membranes associated with the Golgi complex [22,59], although mitochondrial expression has also been reported [60]. NCKX5 expression was originally thought to be restricted to skin melanocytes. However, it has also been found expressed in the brain in the pigmented epithelium of the retina and in the choroid plexus [20,61]. The choroid plexus is a secretory organ found within the ventricles of the brain which produces cerebrospinal fluid [62]. It consists of a core of leaky capillaries and connective tissue surrounded by a tight epithelial monolayer, contiguous with the ependymal cells that line the ventricles. The choroid plexus epithelial cells are responsible for fluid and solute secretion into the cerebrospinal spaces. While salt and water transport are quite well understood here, little is known about the mechanisms which secrete Ca2+ across this epithelial cell layer. The presence of NCKX5 in these cells, presumably in intracellular organelles, suggests that Ca2+ may move through intracellular vesicles across the epithelial layer [62]. Similarly unconventional, organellar, models for Ca2+ transport have been proposed for dental ameloblasts and mammary gland epithelial cells [63,64]. This is a fascinating area of research that is currently under-explored.

10. NCKX genes and human disease Despite their essential and distinct contributions to neuronal Ca2+ homeostasis, NCKX genes have in general not been linked with human disease. This might be as a consequence of homeostatic compensation in Ca2+ signaling brought about by other Ca2+ transporters, including other NCKX family members. Alternatively, it is possible that mutation in a gene so essential for a highly sensitive cellular signaling pathway leads to embryonic lethality, and hence an absence of such alleles from the population. The exceptions to this are the mutation in NCKX1 (SLC24A1) which causes CSNB, as mentioned above [31,71]; mutations in NCKX4 (SLC24A4) which cause amelogenesis imperfecta (AI), a disease in which tooth enamel is defective [55,72–74]; and mutations in NCKX5 (SLC24A5) which cause oculocutaneous albinism (OCA) type 6, a severe hypopigmentation condition [75]. As described above, NCKX1 (SLC24A1) mutations have been associated with the non-degenerative retinal disease, CSNB [31,71,76]. A mouse Nckx1 gene knockout model has also been developed which both recapitulates the human disease and helps explain the mechanism behind the nature of the pathology [32]. It was observed that in these animals there is a compensatory reduction in rod photoreceptor CNG channel density and function, reducing dark-mediated Ca2+ entry, increased Ca2+ efflux via a non-NCKX1 pathway, and an alteration in cellular morphology. These changes appear both to reduce rod function during low light, and allow Ca2+ to be maintained within a range that does not lead to cellular degeneration. Although no mutations have been identified in human SLC24A2, coding for NCKX2, there have been reports that conditions in which neuronal development is altered are associated with changes in expression of the Nckx2 gene in animal models [77,78]. Such changes

9. Regulation of NCKX function Regulation of NCKX function by either substrate availability or allosteric mechanisms is an important, but relatively under-explored area. The direction of NCKX-driven Ca2+ transport is a thermodynamic property that depends only on the electrochemical gradients of Na+, K+, and Ca2+, and the stoichiometry with which these ions bind and are transported. The rate of transport, on the other hand, particularly under basal conditions, is kinetically limited by ion binding, especially by cytoplasmic Ca2+, to the transport sites. Thus, NCKX activity increases as [Ca2+] increases during a signaling event. An additional layer of substrate-dependent activity modulation may be mediated by [K+]. NCKX3 and NCKX4, but not NCKX2, have apparent affinities for extracellular K+ close to typical extracellular concentration values for this ion, so that any increase – for instance due to opening of close-by K+-channels – might lead to kinetic slowing of Ca2+ efflux [65]. Modulation by this means might be particularly important during sustained neuronal activity leading to synaptic plasticity. 6

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would be consistent with the reduction in neuronal Ca2+ flux and alteration in synaptic plasticity and kinase pathways observed in the Nckx2 knockout mice [35,40]. Similarly, no disease mutations have been identified in SLC24A3, coding for NCKX3. Isolated studies have found an association between the Slc24a3 locus and the control of blood glucose in a mouse obesity model [79], as well as an association between a SNP in SLC24A3 and salt-sensitive hypertension [80]. However, no confirmation of these associations in other populations has been reported. Several different mutations in the SLC24A4 gene have all been independently linked to the enamel mineralization disease, amelogenesis imperfecta (AI) [55,72–74]. In all cases, these changes result in a defective NCKX4 protein product [81]. Since NCKX4 has been demonstrated on the apical surface of the maturation stage ameloblasts [54,56], it is likely that lack of Ca2+ extrusion from these cells in the individuals with NCKX4 mutations prevents proper deposition of dental enamel. Perhaps surprisingly, given that animal models in which Nckx4 is knocked out display both olfactory and satiety deficits in addition to the defect in dental enamel production [52,53,55], the human AI patients were not reported to display any other overt health issues beyond the dental ones. Several studies have linked SNPs within the SLC24A4 locus to fair hair and light eyes [82–84]. Furthermore, a number of studies have also linked both SLC24A4 SNPs and altered epigenetic marks to Alzheimer Disease and cognitive aging (reviewed in [85] and [86]). However, the AI patients were not reported to display altered hair or eye colour, nor any signs of cognitive impairment or dementia. It is thus possible that, rather than inducing a change in NCKX4 expression and function, the SNPs and epigenetic marks associated with the SLC24A4 locus are in linkage disequilibrium with another nearby locus causally related to the phenotypes. SLC24A5 was the first of the NCKX genes to be linked to human phenotypic variation, having a clearly defined contribution to skin colour and pigmentation [20]. Moreover, additional mutations in SLC25A5 have been linked to OCA type 6 [75,87,88]. Evidence has been presented which suggests all these mutations result in an NCKX5 protein with either reduced or absent function [81]. Nevertheless, a functional role for the NCKX5 protein and NCKX5-mediated Ca2+ flux in normal melanosome maturation and pigment development has not been established [89]. So far, no neurological symptoms have been connected to any of the mutations in NCKX5.

Acknowledgments The work in the authors' laboratory has been supported by research operating grants from the Canadian Institutes of Health Research and the Natural Sciences & Engineering Research Council of Canada. MTH was supported by a doctoral scholarship from the Libin Cardiovascular Institute of Alberta. References [1] M.J. Berridge, Calcium signalling remodelling and disease, Biochem. Soc. Trans. 40 (2012) 297–309. [2] M. Brini, T. Calì, D. Ottolini, E. Carafoli, Neuronal calcium signaling: function and dysfunction, Cell. Mol. Life Sci. 71 (2014) 2787–2814. [3] R. Bagur, G. Hajnóczky, Intracellular Ca2+ sensing: its role in calcium homeostasis and signaling, Mol. Cell 66 (2017) 780–788. [4] T. Calì, M. Brini, E. Carafoli, Regulation of cell calcium and role of plasma membrane calcium ATPases, Int. Rev. Cell Mol. Biol. (2017) 259–296. [5] F. Wuytack, L. Raeymaekers, L. Missiaen, Molecular physiology of the SERCA and SPCA pumps, Cell Calcium 32 (2002) 279–305. [6] M.P. 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Ho, K + -dependent Na + /Ca2+ exchange is a major Ca2+ clearance mechanism in axon terminals of rat neurohypophysis, J. Neurosci. 22 (2002) 6891–6899. [28] P.P.M. Schnetkamp, The SLC24 Na+/Ca2+-K+ exchanger family: vision and beyond, Pflugers Arch. 447 (2004) 683–688. [29] J. Chen, M.L. Woodruff, T. Wang, F.A. Concepcion, D. Tranchina, G.L. Fain, Channel

11. Concluding remarks The SLC24 gene family, comprising protein products NCKX1–5, is widely and abundantly present in neurons and other cell types throughout the brain, with distinct and partially overlapping patterns of expression. Clear contributions to Ca2+ flux have been demonstrated for most of the isoforms, which have been linked to distinct physiological functions (Fig. 3). In some cases, a direct contribution to human phenotypes and disease has also been demonstrated. Yet, there is much more to learn about how this intriguing family of Ca2+ transport proteins contributes to neuronal and brain molecular physiology. Thirty years have now elapsed since the first description of NCKX function in the outer segments of rod photoreceptors. Despite much effort and insight gained through many studies during the intervening period, it has been only in the past four years that molecular genetic studies have truly revealed the mechanistic consequences of NCKX1 function to rod physiology. It is perhaps, then, not surprising that our understanding of the other family members, only discovered a decade or so after NCKX1, needs further study. We eagerly anticipate what the next decade holds for NCKX research. CRediT authorship contribution statement Mohamed Tarek Hassan: Writing - review & editing. Jonathan Lytton: Conceptualization, Writing - review & editing. 7

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