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 of S. typhimurium (Gibson et al., 1991). CorA is a member of the Metal Ion Transporter (MIT) family of α-type channels (TC #1.A.35).
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.
The generalized reaction that may be catalyzed by CNNM porters is:
Mg2+ (in) → Mg2+ (out)