CrossTalk opposing view: CNNM proteins are not Na+ /Mg2+ exchangers but Mg2+ transport regulators playing a central role in transepithelial Mg2+ (re)absorption.

Introduction

Magnesium (Mg2+) is indispensable for many physiological processes in the cell impacting organ function (de Baaij et al. 2015). Intracellular concentrations of free Mg2+ are maintained within a narrow range (0.5–1.2 mm) through tightly regulated Mg2+ influx and efflux mechanisms (Ebel & Gunther, 1980). Inhibition of Mg2+ efflux by substitution of extracellular Na+ by choline in mammalian cells indicates that Mg2+ extrusion is Na+ dependent (Guther et al. 1984). Thus, it is postulated that Mg2+ efflux occurs via a Na+/Mg2+ exchanger, this notion being corroborated in a large number of studies in different cell models (Romani, 2007).

Under the premise that differential gene expression is involved in the maintenance of cellular Mg2+ homeostasis, microarray analyses in epithelial cells exposed to low extracellular Mg2+ concentrations have been classically used to designate proteins involved in Mg2+ transport (Quamme, 2010). One of the protein families identified with this approach is the CNNM family. CNNM proteins have been proposed to facilitate epithelial Mg2+ extrusion since at least two members of this family, CNNM2 and CNNM4, localize in the basolateral membrane of epithelial cells where apical‐to‐basolateral Mg2+ transport occurs (Stuiver et al. 2011; Yamazaki et al. 2013). However, in this review of the literature, we will demonstrate that the evidence supporting this hypothesis is controversial and several findings suggest that CNNMs cannot function as Na+/Mg2+ exchangers.

Topology

The CNNM family (CNNM1–4) shares a high homology with the bacterial CorC protein, which is related to the maintenance of Mg2+ and Co2+ homeostasis (Hmiel et al. 1989). However, CorC requires CorA to function as a cation transporter and does not transport Mg2+ itself (Gibson et al. 1991). Within the CNNM family, membrane topology studies have demonstrated that CNNM2 is composed of dimers of three transmembrane domains (Fig. 1) (de Baaij et al. 2012). This number of transmembrane domains is significantly lower than that in typical Mg2+ channels/transporters such as TRPM6/M7 (tetramers of 6 transmembrane domains), or SLC41A1/A3 (dimers of 11 transmembrane domains) (Voets et al. 2004; Bates‐Withers et al. 2011; Kolisek et al. 2012; de Baaij et al. 2016). Additionally, a putative pore facilitating cation transport is unlikely to be present in CNNM2 since the re‐entrant loop in its tertiary structure does not completely span the cell membrane and therefore does not facilitate the formation of a typical pore (Fig. 1) (de Baaij et al. 2012).

Figure 1

Membrane topology and subcellular localization of CNNM2

The left panel indicates the membrane topology of CNNM2 showing three full membrane‐spanning domains and a large intracellular C‐terminus containing two CBS modules that bind Mg‐ATP. Reported mutations have been indicated by purple dots. The right panel shows the basolateral localization of CNNM2 in the distal convoluted tubule kidney cell.

A common feature of CNNM proteins is the presence of intracellular cystathionine β‐synthase (CBS) domains (de Baaij et al. 2012). The CBS domains are the main site of regulation, as has recently been demonstrated by a number studies showing the interaction with phosphatases of the regenerating liver (PRL) proteins (Hardy et al. 2015; Gulerez et al. 2016; Kostantin et al. 2016). CBS domains regulate protein function upon binding Mg‐ATP (Corral‐Rodriguez et al. 2014; Hirata et al. 2014). Most intracellular Mg2+ is bound to ATP. Intracellular Mg‐ATP changes in concentration can be sensed by CNNM proteins and may regulate their function(s). Although the exact role between ATP production and Mg2+ concentrations is unknown, an interesting hypothesis is that CNNM proteins function as indirect sensors of intracellular Mg2+ concentrations.

Function

Since its identification in 2003, the function of CNNM proteins has been subject to debate (Wang et al. 2003). Because of their homology to cyclin proteins, CNNMs were initially hypothesized to have a role in cell cycle progression. However, a cyclin function was never demonstrated. For years, CNNM2 has been the main focus of functional CNNM experiments, as the protein was identified in renal distal convoluted tubule (DCT) cells (Goytain & Quamme, 2005; Stuiver et al. 2011; de Baaij et al. 2012). In the DCT, Mg2+ is actively reabsorbed and urinary Mg2+ excretion is determined. In human studies, TRPM6 has been established as the Mg2+ uptake mechanism in this nephron segment (Schlingmann et al. 2002). However, recent studies in mice show that TRPM7 may be equally important in determining renal Mg2+ excretion (Chubanov et al. 2016). The protein mediating Mg2+ extrusion still has to be identified.

As a result, several groups have examined the potential Mg2+ transport properties of CNNM2 using electrophysiology in a multitude of cell models. In 2005, Quamme and his team showed CNNM2‐mediated Mg2+ currents in Xenopus laevis oocytes with a K m for Mg2+ of 0.56 ± 0.05, establishing CNNM2 as a Mg2+ transporter (Goytain & Quamme, 2005). In the DCT, luminal Mg2+ concentrations range between 0.2 and 0.7 mm Mg2+ (Dai et al. 2001), supporting CNNM2‐mediated Mg2+ uptake. However, in mammalian cells, these findings could not be reproduced by two independent research groups (Stuiver et al. 2011; Sponder et al. 2016), although a minor Mg2+‐dependent Na+ current was detected in HEK293 cells overexpressing CNNM2 (Stuiver et al. 2011; Yamazaki et al. 2013). From 2013 onwards, CNNM4 function has also been examined, as it was shown to be expressed in the colon and to be involved in cancer progression (Yamazaki et al. 2013; Funato et al. 2014). In electrophysiological examinations of CNNM4‐expressing cells, no Mg2+ currents were detected (Yamazaki et al. 2013). Because electrophysiology does not permit the detection of electroneutral Mg2+ transport, this led several authors to suggest that CNNM proteins might be Na+‐dependent Mg2+ exchangers at a 2:1 (Na+:Mg2+) ratio.

To tackle this hypothesis, alternative experiments were staged. Several groups have aimed to measure CNNM activity using fluorescent Mg2+ probes, resulting in a large pile of conflicting data. First, using the Mg2+‐sensitive fluorescent Magnesium Green indicator, the group of Miki showed that overexpression of CNNM4 or CNNM2 decreased cellular Mg2+ concentrations (Yamazaki et al. 2013; Funato et al. 2014). Their data suggest that the Mg2+ efflux is Na+ dependent, supporting the exchange theory. Second, Tremblay and colleagues demonstrated CNNM3‐dependent Mg2+ uptake using Mag‐Fura‐2 (Hardy et al. 2015; Kostantin et al. 2016). As their experiments are performed in a physiological 140 mm Na+‐containing buffer, this contradicts Na+ dependency. Third, Kolisek's group could not detect CNNM2‐mediated Mg2+ influx nor efflux, questioning the ability of CNNMs to transport Mg2+ (Sponder et al. 2016). Several important limitations of these experiments have to be considered. All experiments were performed in non‐physiological Mg2+ concentrations, including loading steps using 10 mm up to 40 mm Mg2+. Moreover, the Mg2+ probes are notoriously difficult to work with as they have dissociation constants above physiological Mg2+ concentrations, as well as sensitivity to Ca2+ (Szmacinski & Lakowicz, 1996). Additionally, Magnesium Green is a non‐ratiometric probe, which means that the experiments are extremely sensitive to osmolality changes.

To overcome these hurdles, we have recently developed a stable isotope assay using 25Mg2+ and mass spectrometry. This set‐up measures cellular Mg2+ uptake using the physiological condition of 1 mm Mg2+. Our 25Mg2+ uptake experiments, performed in HEK293 cells overexpressing CNNM2, demonstrate that CNNM2 increases cellular Mg2+ uptake rather than Mg2+ efflux (Arjona et al. 2014). This conclusion is illustrated by the fact that inhibition of Mg2+ efflux through Quinidin, an inhibitor of Na+/Mg2+ exchange, did not affect CNNM2‐mediated 25Mg2+ cellular accumulation. Interestingly, this uptake is independent of Na+, as replacing Na+ with NMDG had no effect, nor did ouabain, an inhibitor of the Na+/K+‐ATPase (Arjona et al. 2014). However, the CNNM2‐dependent Mg2+ uptake was completely abolished by using 2‐aminoethoxydiphenyl borate (2‐APB), which is a known inhibitor of TRPM7 (Chokshi et al. 2012; Arjona et al. 2014).

Altogether, the functional experiments in a wide range of models and techniques show that CNNM2 is not a Na+/Mg2+‐exchanger, as the Mg2+ transport is independent of Na+ and can be bi‐directional (influx and efflux). Moreover, the discrepancy between the different models shows that CNNM2 function is largely dependent on other proteins present in the cell. This is suggestive of a regulating function rather than a transport function. Given that the CNNM2‐dependent 25Mg2+ uptake could be blocked by 2‐APB, TRPM7, a channel involved in Mg2+ homeostasis, is an interesting candidate for regulation by CNNM2 (Paravicini et al. 2012).

In an in vivo context, dysfunctional CNNM2 has been linked to defects in renal Mg2+ reabsorption in mice and zebrafish (Arjona et al. 2014; Funato et al. 2017). In addition, CNNM4 knockout in mice results in impaired intestinal Mg2+ absorption (Yamazaki et al. 2013). Thus, though CNNM proteins are not Mg2+ transporters, they regulate the body Mg2+ balance.

Conclusion and perspective

Hence, the following evidence supports that CNNM proteins are not Na+/Mg2+ exchangers: (i) CNNM proteins do not comply with the consensual topological attributes of a typical Mg2+ transporter, i.e. abundant transmembrane domains and a cation pore formed by membrane‐spanning domains; (ii) conflicting functional data of CNNM‐mediated Mg2+ extrusion between different cellular models indicate that the Mg2+ extrusion function of CNNM proteins depends on the Mg2+ channels/transporters specifically expressed by each cellular model; and (iii) CNNM2‐mediated Mg2+ extrusion in epithelial cells is not dependent on Na+ in a series of experiments. Altogether, this shows that CNNM proteins are not Na+/Mg2+ exchangers. Future research should focus on the identification of the protein partners of CNNMs to elucidate the CNNM‐dependent regulation of Mg2+ transport.

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Additional information

Competing interests

None declared.

Author contributions

Both authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was financially supported by grants from the Netherlands Organization for Scientific Research (NWO VENI 016.186.012) and the Dutch Kidney Foundation (Kolff 14OKG17).