Cross‐talk between zinc and calcium regulates ion transport: A role for the zinc receptor, ZnR/GPR39

Zinc is essential for many physiological functions, with a major role in digestive system, skin health, and learning and memory. On the cellular level, zinc is involved in cell proliferation and cell death. A selective zinc sensing receptor, ZnR/GPR39 is a Gq‐coupled receptor that acts via the inositol trisphosphate pathway to release intracellular Ca2+. The ZnR/GPR39 serves as a mediator between extracellular changes in Zn2+ concentration and cellular Ca2+ signalling. This signalling pathway regulates ion transporters activity and thereby controls the formation of transepithelial gradients or neuronal membrane potential, which play a fundamental role in the physiological function of these tissues. This review focuses on the role of Ca2+ signalling, and specifically ZnR/GPR39, with respect to the regulation of the Na+/H+ exchanger, NHE1, and of the K+/Cl− cotransporters, KCC1‐3, and also describes the physiological implications of this regulation.


Zinc and calcium interactions in cellular signalling
Zinc and calcium cations are essential for numerous cellular functions and they share interesting similarities in their signalling roles and distribution.Both cations are found in steep concentration gradients across the plasma membrane and both are sequestered into cellular organelles.Transient increases in cytoplasmic calcium level, induced by its release from the endoplasmic reticulum (ER) or its influx into the cell, trigger downstream signalling regulating almost all physiological processes, ranging from synaptic transmission to muscle activity and epithelial secretion (Ahuja et al., 2020;Berridge, 2014;Tavi & Westerblad, 2011).Aberrant calcium signalling, on the other hand, is a hallmark of tumour progression, stroke or neurodegenerative diseases (Baev et al., 2022;Iamshanova et al., 2017;Stanisz et al., 2016;Zhang et al., 2019;Zündorf & Reiser, 2011).At the cellular level, calcium is a well-established player in numerous signalling pathways, which include among them those linked to cell proliferation and survival.
Zinc is the second most abundant mineral, after iron, with ∼2 g in the adult human body, and a dietary requirement for 15-25 mg day -1 (Chasapis et al., 2012;Hall & King, 2023).For many years, zinc ions were considered structural elements in as many as 10% of human proteins, interacting with zinc finger domains and serving as enzyme cofactors (Maret, 2013).The concentration of free ionic Zn 2+ is in the picomolar range in both the intracellular and extracellular regions, and it is rapidly bound to proteins that buffer its concentrations, such as metallothioneins or citrate (Colvin et al., 2010;Krężel & Maret, 2016;Maret, 2008).Further studies identified more than 20 Zn 2+ transporter proteins that control levels of free Zn 2+ ions within the cytoplasm and sequester this metal ion into vesicles and distinct organelles (Kambe et al., 2021).Importantly, it is now established that physiological processes can induce transient changes in the level of cytosolic Zn 2+ , similar to Ca 2+ transients.These cytosolic Zn 2+ transients can be mediated, for example, by ZIP proteins that transport Zn 2+ from the extracellular milieu or from cellular organelles (Maret, 2017;Taylor et al., 2012) or following redox activity that releases Zn 2+ from metallothioneins (Zhang et al., 2004).Transient increases in cytosolic Zn 2+ modulate cellular signalling by directly interacting with numerous cellular kinases and phosphatases (Bellomo et al., 2016).
Zn 2+ acts as a first messenger.Surprisingly, Zn 2+ that is not produced or degraded in the cell also acts as a first messenger (Maret, 2017).For this signalling role, free Zn 2+ levels are maintained low in the extracellular space by the ZIP Zn 2+ transporters, as well as by Zn 2+ binding proteins (Kambe et al., 2021;Levaot & Hershfinkel, 2018).
Signalling by Ca 2+ and Zn 2+ ions regulate cell function, survival and death.Among the prominent cellular functions is the activity of ion transporters that maintain ionic gradients across the plasma membrane.These gradients drive solute transport in epithelial cells and modulate neurotransmission, and breakdown of the ionic gradients leads to disease.This review focuses on the general role of Ca 2+ signalling, and specifically that triggered by Zn 2+ , with respect to the regulation of two major ion transporter families: the Na + /H + exchangers and the K + /Cl − cotransporters.
Zn 2+ -dependent Ca 2+ signalling via a selective G-protein coupled ZnR/GPR39.The ZnR/GPR39 mediates between extracellular Zn 2+ transients and intracellular Ca 2+ signalling.The function of ZnR/GPR39 was initially identified using Ca 2+ imaging, in epithelial cells that exhibited Zn 2+ -dependent intracellular Ca 2+ release (Hershfinkel et al., 2001).This response is triggered by 50-200 μm Zn 2+ , and results in a typical metabotropic response that lasts 1-2 min in different tissues, with dramatically high affinity in the nanomolar range in skin keratinocytes (Azriel-Tamir et al., 2004;Besser et al., 2009;Hershfinkel, 2018;Hershfinkel et al., 2001;Sharir et al., 2010).Selective inhibitors that attenuate the IP3-dependent opening of Ca 2+ channels on the ER indicated that extracellular Zn 2+ induces Gq and the IP3-dependent increase in intracellular Ca 2+ (Fig. 1).The ZnR/GPR39 is selectively activated by a transient increase in extracellular Zn 2+ , but not other metal ions (Hershfinkel et al., 2001).The Zn 2+ binding site is composed of two histidine residues and one aspartate, which together form a selective binding site for this ion (Popovics & Stewart, 2011).Histidine residues also serve as pH sensors and indeed Zn 2+ binding to ZnR/GPR39 is impaired by extracellular acidification (Cohen, Asraf et al., 2012), suggesting a complex physiological regulation.Although Zn 2+ can permeate into cells via various Zn 2+ transporters, activation of ZnR/GPR39 is not associated with permeation of this ion into the cells.The fact that Zn 2+ is not rapidly degraded suggests that ZnR/GPR39 can induce extensive and prolonged Ca 2+ surges.A hallmark of Gq-coupled receptors is their desensitization (Kovacs et al., 2009;Pierce & Lefkowitz, 2001), which protects cells from this process, and ZnR/GPR39 indeed undergoes rapid desensitization following exposure of cells to Zn 2+ (Azriel-Tamir et al., 2004;Besser et al., 2009;Sharir et al., 2010).Thus, ZnR/GPR39 is a distinct target adapted to respond to transient changes in extracellular Zn 2+ .
The physiological relevance of ZnR/GPR39 requires that it is located in regions where transient Zn 2+ changes occur.Indeed, a functional ZnR/GPR39 was identified in colon epithelial cells (colonocytes), skin cells (keratinocytes), salivary gland epithelial cell and neurons (Hershfinkel, 2018).All these tissues contain cells that sequester Zn 2+ into vesicles, which are subsequently released during physiological activity.In addition, ZnR/GPR39 was also found in prostate cancer cells and breast cancer cells (Dubi et al., 2008;Ventura-Bixenshpaner et al., 2018), which exhibit aberrant Zn 2+ transporter expression and changes in the tissue Zn 2+ pools (Alam & Kelleher, 2012;Costello & Franklin, 2017).Hence, ZnR/GPR39 is positioned to respond to changes in extracellular Zn 2+ concentration and trigger signalling that subsequently controls cellular functions.Indeed, ZnR/GPR39 regulates ion transporters that have fundamental functions in the tissues where it is expressed.As such, regulation of ion transporters by ZnR/GPR39 establishes gradients for movement of solutes across epithelial barriers, whereas, in neurons, it modulates neurotransmission, as subsequently described below.
Function and regulation of Na + /H + exchangers Activity of ZnR/GPR39 was initially associated with recovery of intracellular pH in HT29 colon epithelial cells (Hershfinkel et al., 2001).Not only is cellular pH a crucial homeostatic factor in controlling solute transport, but also it regulates cellular proliferation and survival (Donowitz et al., 2013;Orlowski & Grinstein, 2004).The Na + /H + exchangers (NHE), which utilize the large Na + gradients across cellular membranes to drive H + efflux, are responsible for recovery of resting intracellular pH following cytosolic acidification (Orlowski & Grinstein, 2011).The Na + /H + exchanger family has 13 members that mediate electroneutral Na + /H + exchange and are expressed on the plasma membrane or intracellular organelles (Donowitz et al., 2013).The ubiquitous NHE1 isoform is activated at low intracellular pH and is selectively inhibited by cariporide (Scholz et al., 1995).Structural analysis indicated the NHE1 has 12 transmembrane domains and a long cytoplasmic C-terminal regulatory domain (Alves et al., 2014).Indeed, modulation of NHE1 activity is mediated by post-translational modification of its C-terminal regulatory domain, in particular by phosphorylation and alterations of NHE1 affinity to H + (Putney et al., 2002).
Ca 2+ signalling involved in the regulation of NHE1.
Activity of ZnR/GPR39 is manifested by a transient intracellular Ca 2+ response (Hershfinkel et al., 2001) and we therefore focus on the role of this pathway in NHE activation.Although intracellular acidification is required for NHE1 activation, various extracellular stimuli, through tyrosine kinases and G-protein coupled receptors, modulate the transport rate of this exchanger (Avkiran & Haworth, 2003;Sardet et al., 1990).The signalling pathways that control NHE1 are mediated by its direct phosphorylation or via regulatory proteins, which induce structural conformational changes that affect the cytosolic facing H + transport site (Fliegel, 2019).Early studies showed that growth factors, such as insulin, platelet-derived growth factor and thrombin, enhance the transport mediated by NHE1 via activation of a Ca 2+ -dependent pathway that is mediated largely by Gαq and Gα 13 (Kitamura et al., 1995).In keratinocytes, Gαq-dependent activation of Ca 2+ signalling by ATP, probably through the purinergic receptors, also enhanced the rate of intracellular pH recovery (Sharir et al., 2010).Endothelin acting via a Gαq-coupled mechanism, which leads to intracellular Ca 2+ release and Rho kinase activation, enhanced NHE1 activity in pulmonary epithelium (Undem et al., 2012).Similar activation of NHE1 by endothelin also plays a role in injury of cardiac cells during ischaemia and reperfusion, presumably by promoting cellular Na + overload (Brunner & Opie, 1998).
Activation of ZnR/GPR39 by transient exposure to Zn 2+ dramatically enhances the recovery rate of intracellular pH in neurons, colonocytes and keratinocytes (Azriel-Tamir et al., 2004;Ganay et al., 2015;Sharir et al., 2010).Several members of the NHE family are functional in epithelial cells and neurons, but ZnR/GPR39-enhanced intracellular pH recovery was mainly mediated by the cariporide-sensitive NHE1 in the colonocytic cell line HT29 and in HaCaT skin keratinocytes (Azriel-Tamir et al., 2004;Sharir et al., 2010).The Zn 2+ -dependent enhancement of NHE1 activity required increases in intracellular Ca 2+ and subsequent activation of ERK1/2 (Fig. 1).Although inhibition of ERK1/2 phosphorylation, by U0126, reversed NHE1 activation by ZnR/GPR39, it is still not known whether this is mediated by direct phosphorylation of NHE1 via this kinase and the exact phosphorylation site(s) on NHE1 were not identified.
Indeed, a major pathway that is involved in the regulation of NHE1 by growth factors is the extracellular signal-regulated kinase (ERK)1/2-mitogen-activated protein kinase (MAPK) pathway (Bianchini et al., 1997).Several independent ERK1/2 phosphorylation sites on NHE1 were identified in cardiomyocytes, where its phosphorylation triggers structural changes of the regulatory domain of NHE1, thereby altering its activity (Fliegel, 2019).Interestingly, ERK1/2 not only directly interacts with NHE1, but also increased NHE1 activity may instead be mediated by downstream p90RSK phosphorylation of serine 703 on NHE1 (Moor & Fliegel, 1999;Takahashi et al., 1999).On the other hand, activation of NHE1 by the tyrosine kinase growth factors epidermal growth factor and platelet-derived growth factor involves protein kinase C (PKC) (Ma et al., 1994).Similarly, PKC mediates activation of NHE1 by several Gq-coupled receptors, including vasopressin, bombesin and α1-adrenergic receptors (Putney et al., 2002).
Further studies identified interaction with Ca 2+ binding proteins that affect NHE1 activity.For example, Akt-dependent phosphorylation of serine 648 on NHE1 modulates the binding site of Ca 2+ -bound calmodulin to the exchanger and thereby enhances its activity (Snabaitis et al., 2008).Regulation of NHE1-dependent transport is also achieved by its stabilization in the cell membrane, which can be mediated by interaction of NHE1 with the Ca 2+ -binding protein calcineurin homologous protein group, playing a role in tumour cell survival (Pang et al., 2002).Moreover, interaction of NHE1 with actin filaments through binding of the anchoring ezrin-radixin-moesin proteins not only localized NHE1 onto the cell membrane, but also was associated with regulation of cell shape and survival (Baumgartner et al., 2004;Wu et al., 2004).

Physiological relevance of regulation of NHE by ZnR/GPR39
Epithelial physiology.In epithelial physiology, changes in pH play a prominent role in the intestinal lumen where mucosal bicarbonate secretion and bacterial fermentation of fatty acids dynamically affect local pH that varies between 5 and 7.5 (Nugent et al., 2001).In addition, the critical function of the epidermal barrier in the skin involves acidic pH levels of ∼5 at the apical layer (Putney et al., 2002).As such, colonocytes and keratinocytes are exposed to changes that affect intracellular pH and indeed NHE activity plays important roles in these cells, maintaining a tight balance between the intracellular and extracellular environment (Donowitz et al., 2013).In addition, NHE1 is often expressed on the basolateral side of epithelial cells, controlling cellular pH and thereby cell growth and elongation that are required for example for epithelial regeneration and cell migration (Pedersen & Counillon, 2019).The activation of the epithelial ZnR/GPR39 enhanced NHE-dependent recovery of intracellular pH in colonocytes and keratinocytes (Fig. 2), which was dependent on ERK1/2 activation (Azriel-Tamir et al., 2004;Sharir et al., 2010).Interestingly, inhibition of NHE activity was not directly associated with ZnR/GPR39-dependent enhanced cell growth as monitored using a scratch assay for keratinocyte proliferation or increased survival of colonocytes treated with butyrate (Cohen, Azriel-Tamir et al., 2012).
Because NHE1 mediates H + efflux, in addition to the recovery of the intracellular pH, NHE1 can also serve as a modulator of extracellular pH.In the skin keratinocytes, this function may contribute to lowering the extracellular pH that determines the skin permeability barrier and thereby induces antimicrobial activity (Elias, 2007), which was previously associated with the role of zinc supplementation to healing of the skin.Interestingly, the levels of zinc are decreased with ageing (Baarz & Rink, 2022;Mocchegiani et al., 2013) and this is concomitant with increases in pH level in the skin and impaired epidermal barrier activity (Rinnerthaler & Richter, 2018).
Similar effects on local extracellular pH may have an important role in the barrier function of the intestinal tissue, where ZnR/GPR39 activity rescues colonocytes from short-chain acid-dependent cellular acidification (Azriel-Tamir et al., 2004;Cohen, Asraf et al., 2012).Furthermore, loss of ZnR/GPR39 resulted in decreased expression of the tight junction protein zonula-1 (Cohen et al., 2014), although whether this is linked to reduced regulation of NHE is not known.A link between NHE and tight junction formation has been directly shown in mice lacking NHE2 that demonstrate reduced expression of tight junction proteins and barrier function (Moeser et al., 2008).

Neuronal
physiology.Particularly sensitive to acidification are neurons that are exposed to changes in intracellular pH during glutamate signalling (Chesler & Kaila, 1992).In addition, hypoxic acidification during excitotoxic activity is a major cause of neuronal demise (Hartley & Dubinsky, 1993;Lee & Jung, 2017).In neurons, NHE1 plays an important role in maintaining normal pH and protecting neurons from acidosis associated with ischaemia hypoxia or other forms of brain injury (Kersh et al., 2009;Uria-Avellanal & Robertson, 2014).Activation of ZnR/GPR39-dependent Ca 2+ signalling increased the activity of NHE and enhanced recovery from acidic pH in hippocampal neuronal cultures (Ganay et al., 2015).This was dependent on ERK1/2 activation and the inhibition of this pathway using U0126 abolished regulation of NHE by ZnR/GPR39.
Changes in extracellular pH levels, however, regulate the ZnR/GPR39 response to Zn 2+ (Cohen, Asraf et al., 2012).As such, the Ca 2+ signalling that is triggered by extracellular Zn 2+ in colonocytes and neurons is diminished when the pH of the extracellular milieu decreases to 6 (Cohen, Asraf et al., 2012;Ganay et al., 2015).The Zn 2+ -binding site on ZnR/GPR39 is composed of two histidine residues, His17 and His19 (Popovics & Stewart, 2011), which may be affected by physiological pH changes considering that the pKa of His residues is ∼6.Surprisingly, the pH sensitivity of ZnR/GPR39 was largely dependent on an Asp residue that is also a part of the Zn 2+ binding site on the receptor (Cohen, Asraf et al., 2012).Thus, at acidic extracellular pH, ZnR/GPR39 fails to increase NHE activity.During acidosis, activation of NHE1, which induces further efflux of H + from cells, can exacerbate the extracellular acidification.Thus, inhibition of ZnR/GPR39, which in turn will block NHE1 activation, can have a protective role by mitigating further NHE1-dependent extracellular acidification (Ganay et al., 2015).Considering the small extracellular space in the brain, this pathway may play a particularly important role rescuing neurons from exacerbating acidosis during ischaemic conditions (Manhas et al., 2010;Wang et al., 2008).
Zinc deficiency is associated mainly with diarrhoea and seizure (Levaot & Hershfinkel, 2018) and, in both of these syndromes, the loss of Cl − gradients is crucial for disease symptoms.Transport of Cl − plays an important role in maintaining cell volume.In the digestive system, Cl − absorption is essential for regulation of cell volume and for the electrolyte balance that supports water absorption; indeed, loss of Cl − is closely associated with diarrhoea (Keely & Barrett, 2022).In neurons, the Cl − gradient supports GABA A -dependent inhibitory function and is therefore essential for maintaining inhibitory-excitatory balance (Hamze et al., 2021;Kahle et al., 2015).The solute carrier 12 family of transporters mediate Cl − transport using the electrochemical gradient of Na + or K + that is established by the Na + /K + ATPase (Gamba, 2005a;Lauf & Adragna, 2000).The SLC12A4-7 genes encode the Na + -independent K + /Cl − cotransporters KCC1-4, which primarily mediate K + -dependent Cl − efflux.These transporters are differentially expressed: KCC1 is the ubiquitous member that controls cell volume, KCC2 is the neuronal isoform, and KCC3 and KCC4 are expressed mostly in neurons and kidney epithelium where they regulate ion reabsorption (Hebert et al., 2004).

Signalling pathways that regulate KCC function.
Members of the KCC family, as well as the Na + -dependent NKCC, have several sites of post translational modification by kinases, with the most prominent being the SPAK (Ste20/SPS1-related proline-and alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase) families (Piechotta et al., 2002).In general, it is well-accepted that their activity is enhanced by phosphorylation of NKCC and dephosphorylation of KCCs (Medina et al., 2014).In addition, kinases of the WNK (with no lysine/K) family are responsible for phosphorylation of KCC family members and thereby inhibition of the co-transporters under baseline hypotonic conditions (Cruz-Rangel et al., 2011;Kahle et al., 2004;Mercado et al., 2016).This pathway plays an important role in reabsorption of solutes in the kidney and is strongly associated with hypertension (Gamba, 2005b).Zinc-dependent activation of cellular signalling via ZnR/GPR39 regulates activity of the KCC transporters.
In neurons, ZnR/GPR39-dependent release of Ca 2+ enhanced Cl − efflux (Chorin et al., 2011).Inhibition of the Zn 2+ -dependent Ca 2+ rise, using the Gq inhibitor YM-254 890 or the phospholipase C-β inhibitor U73122, abolished the Zn 2+ -dependent increases in Cl − efflux in neurons (Fig. 3).Importantly, this regulation of KCC2 was absent in neurons lacking ZnR/GPR39 or synaptic Zn 2+ , suggesting that the Zn 2+ -dependent activation of Ca 2+ signalling is essential for regulation of the Cl − efflux by KCC2 (Chorin et al., 2011).Surprisingly, regulation of KCC2 function by ZnR/GPR39 was independent of the described phosphorylation sites on this cotransporter.Hence, the mechanism for regulation of Cl − transport in neurons by ZnR/GPR39 is independent of PKC activation or direct phosphorylation of putative sites on KCC2 (Asraf et al., 2022).Interestingly, although ZnR/GPR39 activates distinct members of the KCC family in different tissues, the pathway that mediates this regulation is shared between the neuronal KCC2 and the epithelial KCC3, and is mediated by the SNARE protein SNAP23 (Asraf et al., 2022).The SNARE proteins are critical for trafficking of vesicles and subsequent fusion with target membranes, thus mediating most membrane insertion events (Chen & Scheller, 2001).In neurons, SNAP25 is regulating synaptic vesicles fusion and neurotransmitter release (Rizo, 2022), whereas SNAP23 is associated with the regulation of surface expression of ion channels and receptors (Gu et al., 2016;Suh et al., 2010).The ubiquitously expressed SNAP23 is also linked to vesicle fusion and cytokine release from mast cells (Suzuki & Verma, 2008) or exosome release from cancer cells (Wei et al., 2017).In addition, SNAP23 is regulating membrane expression of epithelial sodium channels (Butterworth et al., 2005) and insulin-dependent membrane insertion of the glucose transporter GLUT4 (Kawanishi et al., 2000).The ZnR/GPR39-dependent regulation of SNAP23 is shared in both neurons and epithelial cells, and requires phosphorylation of the IKK-dependent serine 95 and serine 120 on SNAP23 (Asraf et al., 2022).Inhibition of the Ca 2+ signalling or of ERK1/2 completely abolished ZnR/GPR39-dependent regulation of KCC activity (Fig. 2).Moreover, inhibition of the IKK pathway, triggered by Zn 2+ -dependent ERK1/2 phosphorylation of IKK, impaired activation of the neuronal KCC2 (Asraf et al., 2022).In addition, activation of KCC2 in neurons is mediated by direct interaction of the cotransporter with both SNAP23 and syntaxin1A (Asraf et al., 2022;Saadi et al., 2012).The ZnR/GPR39-dependent activation of SNAP23 interaction with KCC2 was mediated by the C-terminal of the transporter, and resulted in increased surface expression of the transporter (Asraf et al., 2022;Chorin et al., 2011;Gilad et al., 2015).

Physiological relevance of regulation of KCC by ZnR/GPR39
Epithelial physiology.Regulation of KCCs is required to modulate absorption or secretion of solutes.A major function of colon epithelial cells (colonocytes) is absorption of Cl − that is coupled to water absorption.Impaired intestinal Cl − homeostasis is a major causative factor of diarrhoea.Activation of ZnR/GPR39-dependent Ca 2+ signalling triggers increased activity of the ubiquitous KCC1 (Fig. 3), expressed on the basolateral membrane of mouse colon epithelial cells (colonocytes), as well as human colonocytes (Sunuwar, Asraf et al., 2017).In breast cancer cells, ZnR/GPR39 activated another member of the KCC family, KCC3 (Chakraborty & Hershfinkel, 2021;Mero et al., 2019).Similar to the pathway activated by Zn 2+ to enhance KCC2 activity, increases in KCC1-or KCC3-dependent transport were abolished in ZnR/GP39 knockout cells and tissues, indicating that KCC family members are regulated by ZnR/GPR39 signalling.Interestingly, expression of KCC1 on the basolateral membrane of colon epithelial cells provides an important pathway for Cl − absorption in the intestine (Fig. 2).
Neuronal physiology.By modulating the Cl − gradients, neuronal NKCC1 and KCC2 are responsible for control of neuronal excitability.ZnR/GPR39-dependent activation of KCC2 (Fig. 2) induced a hyperpolarizing shift in neuronal GABA A signalling (Chorin et al., 2011), suggesting a more inhibitory tone is induced by Zn 2+ via ZnR/GPR39.This may provide an intrinsic homeostatic mechanism for regulation of the excitatory-inhibitory balance because Zn 2+ is co-released with the excitatory glutamate neurotransmitter.Indeed, loss of ZnR/GPR39 resulted in breakdown of this balance and lower susceptibility to kainate induced seizure (Gilad et al., 2015).Interestingly, activation of KCC2 in neurons in the hippocampus is also triggered by Ca 2+ signalling that is mediated by another Gq-protein coupled receptor, the metabotropic glutamate receptor (mGluR) group I receptors (Banke & Gegelashvili, 2008).
By contrast to ZnR/GPR39-dependent activation of KCC2, an increase in intracellular Zn 2+ in neurons is linked to inhibition of KCC2 activity (Hershfinkel et al., 2009;Yassin et al., 2014).These changes are probably mediated by direct interaction of Zn 2+ ions with the transporter and do not involve ZnR/GPR39-dependent Ca 2+ signalling.

ZnR/GPR39 in disease
Zinc deficiency is linked to diarrhoea and bowel diseases, diminished wound healing, increased seizures and mood disorders.Although the molecular links are not well understood, regulation of ion transport by ZnR/GPR39 can provide a mechanism underlying the effects of zinc deficiency in these disease states (Fig. 4).Activation of ZnR/GPR39, and thereby the MAPK pathway, can directly affect cell proliferation or function via pathways that do not involve ion transporters.A controversial role for dietary zinc in these diseases in humans may result from the requirement for proper ZnR/GPR39 activity and not only the presence of Zn 2+ itself.
ZnR/GPR39 in skin and wound healing.The addition of zinc to skin ointments is associated with enhanced wound healing and anti-bacterial mechanisms (Agren, 1990).Activation of ZnR/GPR39 was found in skin epithelial cells, namely keratinocytes (Hershfinkel et al., 2001).The ZnR/GPR39-dependent Ca 2+ signalling was essential for proliferation and migration of the keratinocytic HaCaT cells and thereby enhanced wound healing as measured using the scratch closure assay (Sharir et al., 2010).In addition, Zn/GPR39 enhances intracellular recovery of pH in HaCaT cells via activation of NHE1.Because NHE1 mediates H + efflux, in addition to the recovery of the intracellular pH, NHE1 can also serve as a modulator of extracellular pH.In the skin keratinocytes, this function may contribute to lowering the extracellular pH that determines the skin permeability barrier and thereby induces antimicrobial activity (Elias, 2007), which was previously associated with the role of zinc supplementation to healing of the skin.Interestingly, the levels of zinc are decreased with ageing (Baarz & Rink, 2022;Mocchegiani et al., 2013), and this is concomitant with increases in pH level in the skin and impaired epidermal barrier activity (Rinnerthaler & Richter, 2018).

The anti-diarrheal role of
ZnR/GPR39.Zinc supplementation is recommended by the World Health Organization as an effective method to treat diarrhoea (Lamberti et al., 2013;Lazzerini & Wanzira, 2016;Li et al., 2022).Water loss in diarrhoea is accompanied by loss of Na + and Cl − into the lumen (Keely & Barrett, 2022;Singh et al., 2014).Activation of ZnR/GPR39 by Zn 2+ in colonocytes enhanced transport activity mediated by both NHE and KCC1 and can therefore play a major role in diarrheal diseases.
Regulation of KCC1 is directly linked to Cl − absorption, which is essential to reduce water loss in the intestine.Immunohistological studies show that KCC1 is the basolateral Cl − transporter in the intestine (Sunuwar, Asraf et al., 2017) and therefore provides a major pathway for absorption of Cl − in the colon.Regulation of KCC1 by luminal Zn 2+ via ZnR/GPR39 provides an important mechanism underlying the observation that dietary Zn 2+ supplementation ameliorates diarrhoea, whereas zinc deficiency worsens it (Sinha et al., 2022;Tsang et al., 2021).Indeed, ZnR/GPR39 knockout mice that were treated with cholera toxin showed enhanced fluid loss compared to wild-type animals expressing ZnR/GPR39.Moreover, dietary Zn 2+ deficiency enhanced water loss in ZnR/GPR39-expressing animals and had no effect of mice lacking ZnR/GPR39.In the intestinal tissue, moreover, NHE1 also affects Na + absorption and plays a direct role in diarrhoea caused by inflammatory or infectious diseases (Gurney et al., 2017;Sullivan et al., 2009;Surawicz, 2010;Zachos et al., 2009).Thus, enhancement of NHE1 activity in the colon epithelial cells by ZnR/GPR39 can provide a mechanism for the beneficial effect of zinc in reducing severity of diarrhoea (Das et al., 2018).Indeed, mice lacking ZnR/GPR39 expression show impaired recovery from diarrhoea associated with experimental colitis (Sunuwar et al., 2016).In the colonocytic HT29 cell line, ZnR/GPR39 increases NHE transport that is mediated by activation NHE1 as shown using selective inhibitors.However, NHE1 is the basolateral transporter that is responsible for Na + influx into the colonocytes (Xu et al., 2018).This in turn may reduce the intercellular Na + loss into the lumen and thereby water loss; indeed, such a mechanism was associated with loss on NHE1 in patients of inflammatory bowel disease (Khan et al., 2003).In addition, NHE1 activity affects cellular pH and may indirectly regulate solute and water absorption to reduce diarrhoea.Further studies are required to test whether ZnR/GPR39 activity in colon tissue can also enhance NHE3-dependent transport that is directly linked to Na + absorption from the lumen, playing a crucial role in diarrhoea (Khan et al., 2003).

Zinc signalling by ZnR/GPR39 and breast cancer.
Dysregulation of Zn 2+ and its transporters was found to correlate with breast cancer subtype and malignancy (Chandler et al., 2016;Farquharson et al., 2009).Changes were also observed in the levels of metallothioneins, Zn 2+ binding proteins, which can affect the levels of this ion in the cytoplasm and thereby multiple kinases and phosphatases (Bellomo et al., 2016).Expression of ZnR/GPR39 is upregulated in ER negative breast cancer cell lines that are linked to more malignant phenotypes (Ventura-Bixenshpaner et al., 2018).Although ZnR/GPR39 activation of MAPK pathway can enhance cell proliferation, in ER negative breast cancer cells, this signalling pathway enhanced KCC3-dependent transport activity (Chakraborty et al., 2021;Mero et al., 2019).Regulation of KCC3 in cancer cells enhanced tumour growth (Chen et al., 2010;Chiu et al., 2014;Mero et al., 2019) and, in cervical cancer cells, was associated with enhanced cell invasion and proliferation (Shen et al., 2000).Increases in KCC3 and KCC4 expression induced by insulin-like growth factor 1 were also linked to breast cancer progression (Hsu et al., 2007).The effects of Zn 2+ on KCC3 activity were dependent on ZnR/GPR39 expression and downstream Ca 2+ signalling (Mero et al., 2019).Importantly, KCC3 was localized to cellular protrusions, probably formed by local perturbations of cell volume mediated by KCC3 activity (Chakraborty et al., 2021).These protrusions can promote the migration of breast cancer cells and, indeed, both ZnR/GPR39 and KCC3 are required to enhance breast cancer cell invasion through Matrigel (Chakraborty & Hershfinkel, 2021;Chakraborty et al., 2021).Importantly, a role for this pathway in breast cancer is also supported by increased expression of ZnR/GPR39 in tissue biopsies of higher-grade compared to lower-grade tumours (Ventura-Bixenshpaner et al., 2018).
The ERK1/2-MAPK pathway is closely associated with cancer cell proliferation, and links NHE1 to enhanced tumour growth (Anjum et al., 2022;Chuderland & Seger, 2005).Activation of NHE1 can also be mediated by the mutated oncogene Ras, which utilizes the downstream ERK1/2 activation to enhance phosphorylation of NHE1, thereby increasing its transport activity (Hagag et al., 1987;Maly et al., 1989).Although ZnR/GPR39 activation of NHE in keratinocytes was not associated with cell proliferation, this was not investigated in breast cancer cells and requires further attention.
The role of ZnR/GPR39 in controlling seizure.An impaired balance between the excitatory and inhibitory neuronal activity underlies seizure activity.Neuronal inhibition relies on the activity of KCC2 that mediates Cl − extrusion and sets the Cl − gradients for hyperpolarizing GABA A currents.Indeed, epileptogenesis and seizures are tightly linked to KCC2 dysfunction (Duy et al., 2019;Medina et al., 2014).This Cl − transporter is regulated by ZnR/GPR39, and Zn 2+ -dependent activation increases its surface expression and thereby activity.As such, ZnR/GPR39 knockout (KO) mice exhibit enhanced susceptibility to kainate-induced seizure, reaching a higher behavioural score and lasting longer periods (Gilad et al., 2015).Kainate is an agonist of a subfamily of glutamate receptors that enhances excitatory transmission, leading to the release of Zn 2+ at similar levels in the brain of ZnR/GPR39 KO or wild-type mice (Gilad et al., 2015).Furthermore, kainate-dependent activation of KCC2 and thereby Cl − efflux is enhanced in wild-type J Physiol 602.8 mice but not in ZnR/GPR39 KO mice.This suggested a mechanism that can underlie the effects of Zn 2+ on seizure, which requires functional ZnR/GPR39 signalling.
Control of KCC2 activity in neurons has been indeed studied as a potential handle for treating seizure (Delpire, 2021;Kaila et al., 2014;Virtanen et al., 2021).Activation of G-protein coupled mGluRs enhanced the activity of the neuronal KCC2 via PKC-dependent phosphorylation of KCC2 on serine 940 (Lee et al., 2007).This phosphorylation of KCC2 enhanced cell membrane insertion and stability of the transporter.Similar regulation of KCC2 surface expression is mediated by dephosphorylation of threonine 1007 and 906 on this protein (Friedel et al., 2015).

Conclusions and future directions
The function of a selective Zn 2+ sensing receptor in epithelial cells and neurons triggers important Ca 2+ signalling and subsequent pathways that modulate essential functions of the cells.Activation of Gq-coupled receptors involves phospholipase C-mediated hydrolysis of the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) that is generating IP3 and diacylglycerol.Although IP3 induces the release of intracellular Ca 2+ , the depletion of PIP 2 can in itself affect function ion transporters such as the Ca 2+ channel, CaV1.2 (Voelker et al., 2023) or the Na + -bicarbonate cotransporter (Thornell et al., 2012).This activity has been linked to modulation of smooth muscle and endothelial cell function (Harraz et al., 2020).Activity of ZnR/GPR39 in endothelial cells has been described previously (Iovino et al., 2022); however, signalling via PIP 2 needs to be further studied.The ZnR/GPR39 signalling pathway ubiquitously triggers phosphorylation of ERK1/2 in normal epithelial cells, cancerous cells or neurons, which leads to the regulation of ion transporters.Further studies are required to understand how the ZnR/GPR39 pathway is activating NHE1 transport, and whether activation of the various Na + , K + or Cl − transporters is mediated by a common pathway.For example, in neurons, ZnR/GPR39 enhances transport mediated by both, KCC2 and NHE1; however, regulation of NHE1 plays an important role following cellular acidification and is refractory under baseline conditions, whereas KCC2 transport is triggered by physiological neuronal activity.For example, it would be important to determine whether these pathways interact during epileptic seizure when KCC2 plays a crucial role in ion homeostasis, although the resulting cellular acidosis may also trigger NHE1 activation.Similarly, acidosis of the tumour microenvironment may trigger NHE1 activity and modulate KCC3 regulation by ZnR/GPR39 in epithelial cells.Altogether, ZnR/GPR39 offers a novel target triggering Ca 2+ signalling and regulates ion transporters that are not only fundamental for cell survival and function, but also associated with neuronal syndromes ranging from ischaemia to epilepsy and cancer.

Figure 2 .
Figure 2. Schematic representation of ZnR/GPR39-dependent Ca 2+ signalling in colonocytes Luminal Zn 2+ changes activate an apical ZnR/GPR39 that induces Ca 2+ release in the colonocytes.This pathway regulates NHE and KCC1 activity and increases tight junction formation.The figures in this manuscript were partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.
Figure 3. Schematic representation of ZnR/GPR39-dependent activation of KCC2Release of synaptic Zn 2+ activates ZnR/GPR39 and subsequently an increase in cytosolic Ca 2+ and ERK1/2 phosphorylation.Further activation of the SNAP23 enhances plasma membrane insertion of KCC2 and enhanced inhibitory drive.The figures in this manuscript were partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

Figure 4 .
Figure 4.A role for ZnR/GPR39 signalling in diseaseZinc plays a well-known role in epithelial and nervous system function; however, the mechanisms are not well understood.In neurons, colonocytes, keratinocytes and cancer cells, a role for ZnR/GPR39 was described and downstream regulation of ion transport mechanisms was identified.In these tissues, loss of ZnR/GPR39 is associated with the diseases indicated.The figures in this manuscript were partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.