1.A.112. The Cyclin M Mg²⁺ Exporter (CNNM) Family

The hemolysin C of Brachyspira hyodysenteriae (268 aas; ter Huurne et al., 1994) and the Co²⁺-resistance protein, CorC of Salmonella typhimurium (273 aas with one putative TMS (residues 163-181)) are homologous throughout most of their lengths to each other. They are also similar to the C-terminal hydrophilic portions of 5 close paralogues in Bacillus subtilis, all of which are about 440 aas long, have an N-terminal 4 TMS domain, and may be Mg²⁺ exporters (Ulens 2018). One representative B. subtilis paralogue is YrkA (434 aas; spP54428). The CorC protein was believed to function as an auxiliary protein to the CorA Co²⁺/Mg²⁺ channel (TC #1.A.35) of S. typhimurium (Gibson et al., 1991). The bacterial CorB/CorC/CNNM sub-family of proteins is involved in resistance to antibiotic exposure and the survival of pathogenic microorganisms in their host environment. CorC proteins possess a cytoplasmic region containing a (regulatory?) ATP-binding site (Huang et al. 2021).

CBS-pair domain divalent metal cation transport mediators (CNNMs) are a class of Mg²⁺ transporters present throughout biology. Originally discovered in bacteria, there are four CNNM proteins in humans, which are involved in divalent cation transport, genetic diseases, and cancer (Chen and Gehring 2023). Eukaryotic CNNMs are composed of four domains: an extracellular domain, a transmembrane domain, a cystathionine-β-synthase (CBS)-pair domain, and a cyclic nucleotide-binding homology domain. The transmembrane and CBS-pair core are the defining features of CNNM proteins.  Chen and Gehring 2023 reviewed the structural and functional studies of eukaryotic and prokaryotic CNNMs that underlie our understanding of their regulation and mechanism of ion transport. Structures of prokaryotic CNNMs confirm that the transmembrane domain mediates ion transport with the CBS-pair domain likely playing a regulatory role through binding divalent cations. Studies of mammalian CNNMs have identified new binding partners.

Mouse ACDP2 (CNNM2) was cloned from mouse distal convoluted tubule cells of the kidney, expressed in Xenopus laevis oocytes, and studied with two-electrode voltage-clamp techniques. When expressed in oocytes, ACDP2 was reported to mediate saturable Mg²⁺ uptake with a Michaelis constant of 0.56 mM. Transport of Mg²⁺ by ACDP2 was rheogenic and voltage-dependent, but was not coupled to Na⁺ or Cl^- ions. ACDP2 transports a range of divalent cations: Mg²⁺ , Co²⁺, Mn²⁺, Sr²⁺, Ba²⁺, Cu²⁺, and Fe²⁺, thus exhibiting wide substrate selectivity. The cations Ca²⁺, Cd²⁺, Zn²⁺, and Ni²⁺ did not induce currents, and only Zn²⁺ effectively inhibited transport. The ACDP2 transcript is abundantly present in kidney, brain, and heart with lower amounts in liver, small intestine, and colon. Moreover, ACDP2 mRNA is upregulated with magnesium deficiency, particularly in the distal convoluted tubule cells, kidney, heart, and brain. Thus, ACDP2 may be a regulated transporter for Mg²⁺ and other divalent cations in epithelial cells (Goytain et al., 2005).

Mutations in the cyclin M2 (CNNM2) gene were identified to be causative for severe hypomagnesemia. In kidney, CNNM2 is a basolaterally expressed protein with predominant expression in the distal convoluted tubule. Transcellular magnesium (Mg²⁺) reabsorption in the distal convoluted tubule represents the final step before Mg²⁺ is excreted into the urine, thus fine-tuning its final excretion via a tightly regulated mechanism. Membrane topology studies showed that CNNM2 has an extracellular N terminus and an intracellular C terminus (de Baaij et al., 2012). The loss-of-function mutation found in patients disturbs ATP binding by the intracellular cystathionine β-synthase domains. In the endoplasmic reticulum, the signal peptidase complex cleaves off a large N-terminal signal peptide of about 64 amino acids. Mutagenesis screening showed that CNNM2 is glycosylated at residue Asn-112, stabilizing CNNM2 on the plasma membrane. CNNM2a forms heterodimers with the smaller isoform CNNM2b.

The bacterial proteins, YrkA and YhdP, have three recognized domains: the 4-TMS DUF21 domain (residues 1-170), a nucleotide binding CBS domain (residues 225-335) and a CorC/HlyC hydrophilic domain (residues 360-430).  The mammalian homologues have at least the first two of these domains which are preceded by an N-terminal TMS and an unidentified hydrophilic domain. The bacterial HlyC and CorC proteins (1.C.126) lack the 4 TMS DUF21 domain, but have the CBS and CorC/HlyC domains. Only the proteins with the DUF21 domain are likely to be transporters.  All of  the evidence is consistent with the conclusion that these homologues form divalent-cation-specific porters or channels.

There is controversy as to (1) whether the CNNM (cyclin) proteins are transporters or regulators, and (2) if they are transporters, whether they are channels or carriers.  The CBS domains bind ATP (Hirata et al. 2014). In humans, the CNNM family is encoded by four genes: CNNM1-4. CNNM1 is thought to act as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg²⁺) wasting, which may lead to symptoms of Mg²⁺ depletion such as tetany, seizures and cardiac arrhythmias (Gómez-García et al. 2012). In marine fish, there are 6 CNNM paralogues, each with a different location and function, probably catayzing Mg²⁺ efflux (Islam et al. 2014). Three conserved dileucine motifs in CNNM4 are necessary for both basolateral sorting and interaction with the μ1As (AP1A) and μ1B (AP1B) proteins (Hirata et al. 2014). Ishii et al. 2016 also concluded that CNNM proteins are efflux permeases. 

PRL phosphatases and CNNM proteins form complexes, regulated by the formation of phosphocysteine. Gulerez et al. 2016 showed that cysteine in the PRL phosphatase catalytic site is endogenously phosphorylated as part of the catalytic cycle and that phosphocysteine levels change in response to Mg²⁺ levels. Phosphorylation blocks PRL binding to the CNNM Mg²⁺ transporters, and mutations that block the PRL-CNNM interaction prevent regulation of Mg²⁺ efflux in cultured cells. The crystal structure of the complex of PRL2 and the CBS-pair domain of the Mg²⁺ transporter CNNM3 revealed the molecular basis for the interaction (Gulerez et al. 2016). The structural basis underlying the interaction between PRL phosphatases and CNNM transporters has been further studied (Giménez-Mascarell et al. 2017). Renal function of CNNM2 is necessary for maintenance of blood pressure (Funato et al. 2017), and Funato et al. 2018. have concluded that CNNM porters are Na⁺:Mg²⁺ antiporters. However Arjona and de Baaij 2018 suggested that they function as regulators of Mg²⁺ transport, although Funato et al. 2018 rebutted this suggestion. Chen et al. 2018 concluded that the CBS domains in CNNM proteins mediate dimerization and are important for Mg²⁺ transport activity. Most of the evnidence therefore suggests that CNNM proteins are Mg²⁺ exporters (Ulens 2018).

The cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) may be Mg²⁺ transporters (Chen et al. 2018). CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-β-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain. Chen et al. 2018 determined the crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. These domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. An inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg²⁺ efflux was noted. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member, CNNM3, was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg²⁺ efflux activity of CNNM4.

CNNM/CorB proteins are a broadly conserved and are associated with Mg²⁺ transport but it was not known if they mediate transport themselves or regulate other transporters. Chen et al. 2021 determined the crystal structure of an archaeal CorB protein in two conformations (apo and Mg²⁺-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg²⁺ ion coordinated by a conserved pi-helix. In the absence of Mg²⁺-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. A role of the structural rearrangements in mediating Mg²⁺-ATP sensing was suggested. An in vitro, liposome-based assay was used to demonstrate direct Mg²⁺ transport by CorB proteins (Chen et al. 2021).  CNNM2 and CNNM4 are found abundantly in the basolateral membrane of kidney and colon epithelial cells, where renal/intestinal (re)absorption of Mg²⁺ occurs. In humans, mutations in CNNM proteins are linked to two genetic diseases: CNNM2 mutations cause hypomagnesemia while mutations in CNNM4 are associated with Jalili syndrome. CNNM2-knockout mice are embryonic lethal, while loss of CNNM4 leads to male infertility and susceptibility to cancer. CNNMs are additionally implicated in hypertension, non-alcoholic steatohepatitis, and schizophrenia (Chen et al. 2021). CorB was proposed to mediate Mg²⁺ efflux together with CorC and CorD, in which CorC is a soluble protein that shares high sequence similarity to the cytosolic domains of CorB. Subsequently, many other CorB orthologs have been implicated in Mg²⁺ transport. For example, the Staphylococcus aureus ortholog, MpfA, is thought to function as a Mg²⁺ exporter as deletion mutants are unable to grow in the presence of high concentrations of magnesium. Disruption of the homologous gene (yhdP) in Bacillus subtilis leads to increased cellular Mg²⁺ content, again supporting a role in Mg²⁺ efflux (Chen et al. 2021).  MtCorB from a thermophilic archaeon (Methanoculleus thermophilus) has been crystalized and structurally illucidated (Chen et al. 2021).

In prokaryotes, cellular Mg²⁺ homeostasis is orchestrated via the CorA, MgtA/B, MgtE, and CorB/C Mg²⁺ transporters (Franken et al. 2022). For CorA, MgtE, and CorB/C, the motifs that form the selectivity pore, are conserved during evolution. Thus, CNNM proteins, the vertebrate orthologues of CorB/C, also have Mg²⁺ transport capacity. Whereas CorA and CorB/C proteins share the gross quaternary structure and functional properties with their respective orthologues, the MgtE channel only shares the selectivity pore with SLC41 Na⁺/Mg²⁺ transporters (Franken et al. 2022).

CNNMs (CBS-pair domain divalent metal cation transport mediators) are a ubituitous family of Mg transporters.  There are four CNNM proteins in humans, which are involved in divalent cation transport, genetic diseases, and cancer (Chen and Gehring 2023).  Eukaryotic CNNMs are composed of four domains: an extracellular domain, a transmembrane domain, a CBS (cystathionine-beta-synthase)-pair domain, and a cyclic nucleotide-binding homology domain. The transmembrane and CBS-pair core are the defining features of CNNM proteins with over 20,000 protein sequences known from over 8,000 species. Chen and Gehring 2023 reviewed the structural and functional studies of eukaryotic and prokaryotic CNNMs that underlie our understanding of the regulation and mechanism of ion transport. Prokaryotic CNNMs indicates that the transmembrane domain mediates ion transport with the CBS-pair domain playing a regulatory role by binding divalent cations (Chen and Gehring 2023).

The generalized reaction that may be catalyzed by CNNM porters is:

Mg²⁺ (in) → Mg²⁺ (out)