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1.C.126 The HlyC  (HlyC) Family of Haemolysins

The putative 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 and to the C-terminal portions of 5 close paralogues in Bacillus subtilis, all of which are about 440 aas long and have an N-terminal 4 TMS domain. One representative B. subtilis paralogue is YrkA (434 aas; P54428). 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). The HlyC family corresponds to SwissProt family UPF0053. MstE (1.A.26.1.2), CLC (2.A.49.6.1) and HlyC/CorC may all share a hydrophilic domain, and members of the HlyC family lack the 4 TMS transmembrane region and therefore are probably not transporters (see below).


The bacterial proteins, YrkA (1.C.126.2.1) and YhdP (1.C.126.2.2) have three recognized domains: the 4-TMS DUF21 domain (residues 1-170), a nucleotide binding CBS domain (residues 225-335) and a CorC/HlyC 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.126.1.1 and 1.C.126.1.2) lack the 4 TMS DUF21 domain, but have the CBS and CorC/HlyC domains.  Functions of the spirochete HlyC and the Salmonella CorC as transporters are in doubt. 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. Interestingly, 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.

This family belongs to the: CorC/HlyC Domain Family.

References associated with 1.C.126 family:

Brandao K., Deason-Towne F., Perraud AL. and Schmitz C. (2013). The role of Mg2+ in immune cells. Immunol Res. 55(1-3):261-9. 22990458
Gibson, M.M., Bagga, D.A., Miller, C.G., and Maguire, M.E. (1991). Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system. Mol Microbiol. 5: 2753-2762. 1779764
Quamme GA. (2010). Molecular identification of ancient and modern mammalian magnesium transporters. Am J Physiol Cell Physiol. 298(3):C407-29. 19940067
Arjona, F.J. and J.H.F. de Baaij. (2018). CrossTalk opposing view: CNNM proteins are not Na /Mg exchangers but Mg transport regulators playing a central role in transepithelial Mg (re)absorption. J. Physiol. 596: 747-750. 29383729
Chen, Y.S., G. Kozlov, R. Fakih, Y. Funato, H. Miki, and K. Gehring. (2018). The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg efflux activity. J. Biol. Chem. [Epub: Ahead of Print] 30341174
Corral-Rodriguez MA., Stuiver M., Abascal-Palacios G., Diercks T., Oyenarte I., Ereno-Orbea J., de Opakua AI., Blanco FJ., Encinar JA., Spiwok V., Terashima H., Accardi A., Muller D. and Martinez-Cruz LA. (2014). Nucleotide binding triggers a conformational change of the CBS module of the magnesium transporter CNNM2 from a twisted towards a flat structure. Biochem J. 464(1):23-34. 25184538
de Baaij, J.H., M. Stuiver, I.C. Meij, S. Lainez, K. Kopplin, H. Venselaar, D. Müller, R.J. Bindels, and J.G. Hoenderop. (2012). Membrane topology and intracellular processing of cyclin M2 (CNNM2). J. Biol. Chem. 287: 13644-13655. 22399287
Funato, Y., D. Yamazaki, and H. Miki. (2017). Renal function of cyclin M2 Mg2+ transporter maintains blood pressure. J Hypertens 35: 585-592. 28033128
Funato, Y., K. Furutani, Y. Kurachi, and H. Miki. (2018). CrossTalk proposal: CNNM proteins are Na /Mg exchangers playing a central role in transepithelial Mg (re)absorption. J. Physiol. 596: 743-746. 29383719
Funato, Y., K. Furutani, Y. Kurachi, and H. Miki. (2018). Rebuttal from Yosuke Funato, Kazuharu Furutani, Yoshihisa Kurachi and Hiroaki Miki. J. Physiol. 596: 751. 29383723
Giménez-Mascarell, P., I. Oyenarte, S. Hardy, T. Breiderhoff, M. Stuiver, E. Kostantin, T. Diercks, A.L. Pey, J. Ereño-Orbea, M.L. Martínez-Chantar, R. Khalaf-Nazzal, F. Claverie-Martin, D. Müller, M.L. Tremblay, and L.A. Martínez-Cruz. (2017). Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2. J. Biol. Chem. 292: 786-801. 27899452
Gómez-García, I., M. Stuiver, J. Ereño, I. Oyenarte, M.A. Corral-Rodríguez, D. Müller, and L.A. Martínez-Cruz. (2012). Purification, crystallization and preliminary crystallographic analysis of the CBS-domain pair of cyclin M2 (CNNM2). Acta Crystallogr Sect F Struct Biol Cryst Commun 68: 1198-1203. 23027747
Goytain, A., and G.A. Quamme. (2005). Functional characterization of ACDP2 (ancient conserved domain protein), a divalent metal transporter. Physiol Genomics. 22: 382-389. 15899945
Gulerez, I., Y. Funato, H. Wu, M. Yang, G. Kozlov, H. Miki, and K. Gehring. (2016). Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis. EMBO Rep 17: 1890-1900. 27856537
Hirata, Y., Y. Funato, and H. Miki. (2014). Basolateral sorting of the Mg²⁺ transporter CNNM4 requires interaction with AP-1A and AP-1B. Biochem. Biophys. Res. Commun. 455: 184-189. 25449265
Hirata, Y., Y. Funato, Y. Takano, and H. Miki. (2014). Mg2+-dependent interactions of ATP with the cystathionine-β-synthase (CBS) domains of a magnesium transporter. J. Biol. Chem. 289: 14731-14739. 24706765
Ishii, T., Y. Funato, O. Hashizume, D. Yamazaki, Y. Hirata, K. Nishiwaki, N. Kono, H. Arai, and H. Miki. (2016). Mg2+ Extrusion from Intestinal Epithelia by CNNM Proteins Is Essential for Gonadogenesis via AMPK-TORC1 Signaling in Caenorhabditis elegans. PLoS Genet 12: e1006276. 27564576
Islam, Z., N. Hayashi, H. Inoue, T. Umezawa, Y. Kimura, H. Doi, M.F. Romero, S. Hirose, and A. Kato. (2014). Identification and lateral membrane localization of cyclin M3, likely to be involved in renal Mg2+ handling in seawater fish. Am. J. Physiol. Regul Integr Comp Physiol 307: R525-537. 24965791
Sałamaszyńska-Guz, A. and D. Klimuszko. (2008). Functional analysis of the Campylobacter jejuni cj0183 and cj0588 genes. Curr. Microbiol. 56: 592-596. 18389311
Schäffers, O.J.M., J.G.J. Hoenderop, R.J.M. Bindels, and J.H.F. de Baaij. (2018). The rise and fall of novel renal magnesium transporters. Am. J. Physiol. Renal Physiol 314: F1027-F1033. 29412701
Simonin A. and Fuster D. (2010). Nedd4-1 and beta-arrestin-1 are key regulators of Na+/H+ exchanger 1 ubiquitylation, endocytosis, and function. J Biol Chem. 285(49):38293-303. 20855896
ter Huurne, A.A., Muir, S., van Houten, M., van der Zeijst, B.A., Gaastra, W., and Kusters, J.G. (1994). Characterization of three putative Serpulina hyodysenteriae hemolysins. Microb Pathog. 16: 269-282. 7968456
Turner, M.S. and J.D. Helmann. (2000). Mutations in multidrug efflux homologs, sugar isomerases, and antimicrobial biosynthesis genes differentially elevate activity of the σX and σW factors in Bacillus subtilis. J. Bacteriol. 182: 5202-5210. 10960106