2.A.97 The Mitochondrial Inner Membrane K⁺/H⁺ and Ca²⁺/H⁺ Exchanger (LetM1) Family Mitochondria are integral components of cellular calcium (Ca²⁺) signaling. Calcium stimulates mitochondrial adenosine 5''-triphosphate production, but can also initiate apoptosis. In turn, cytoplasmic Ca²⁺ concentrations are regulated by mitochondria. Leucine zipper EF hand-containing transmembrane protein 1 (LetM1) and uncoupling proteins 2 and 3 (UCP2/3) contribute to two distinct mitochondrial Ca²⁺ uptake pathways (Waldeck-Weiermair et al., 2011). Lin and Stathopulos 2019 presented an overview of the current understanding of LETM1 structure, mechanism and function. LETM1 is required for mitochondrial homeostasis and cellular viability (Li et al. 2019). The LetM1 family has also been designated the SLC55 family (Gyimesi and Hediger 2020). LetM1 is a large human protein of 739 aas with 1 TMS. It is implicated in the Wolf-Hirschhorn Syndrome. Homologues are found in plants and fungi. These proteins in yeast and humans are in the inner mitochondrial membrane and are complementary. The human protein corrects the yeast defect. Deletion of the yeast protein yields defective, swollen mitochondria with increased cation (K⁺) contents and low Δψ. The primary defect may be in the mitochondrial K⁺/H⁺ exchange activity. These proteins therefore function both in K⁺ homeostasis and organelle volume control (Nowikovsky et al., 2004). Jiang et al. (2009) searched for genes that regulate mitochondrial Ca²⁺ and H⁺ concentrations using a genome-wide Drosophila RNA interference (RNAi) screen. The mammalian homolog of one Drosophila gene identified in the screen, LetM1, was found to specifically mediate coupled Ca²⁺/H⁺ exchange. RNAi knockdown, overexpression, and liposome reconstitution of the purified LetM1 protein demonstrate that LetM1 is a mitochondrial Ca²⁺/H⁺ antiporter (Jiang et al., 2009). This system is discussed by Santo-Domingo and Demaurex (2010). Leucine zipper-EF-hand containing transmembrane protein 1 (LetM1), one of the genes deleted in Wolf-Hirschhorn syndrome, encodes a putative mitochondrial Ca²⁺/H⁺ antiporter. Cellular Letm1 knockdown reduced Ca²⁺mito uptake, H⁺mito extrusion and impaired mitochondrial ATP generation capacity. Homozygous deletion of LetM1 in mice resulted in embryonic lethality before day 6.5 of embryogenesis and ~50% of the heterozygotes died before day 13.5 of embryogenesis (Jiang et al. 2013). The surviving heterozygous mice exhibited altered glucose metabolism, impaired control of brain ATP levels, and increased seizure activity. Thus, loss of LetM1 contributes to the pathology of Wolf-Hirschhorn syndrome in humans and may contribute to seizure phenotypes by reducing glucose oxidation. Purified, and reconstituted human LetM1 exhibits apparent affinities of cations in the order Ca²⁺ = Mn²⁺ > Gd³⁺ = La³⁺ > Sr²⁺ >> Ba²⁺, Mg²⁺, K⁺, Na⁺. Kinetic analyses suggested a LetM1 turnover rate of 2 Ca²⁺/s and a K_m of ~ 25 microM. LetM1 mediates electroneutral 1 Ca²⁺/2 H⁺ antiport. LetM1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na⁺/Ca²⁺exchanger. Functional properties of LetM1 are similar to those of the H⁺-dependent Ca²⁺ transport mechanism identified in intact mitochondria (Tsai et al. 2013).
References:
Alam, M.R., L.N. Groschner, W. Parichatikanond, L. Kuo, A.I. Bondarenko, R. Rost, M. Waldeck-Weiermair, R. Malli, and W.F. Graier. (2012). Mitochondrial Ca²⁺ uptake 1 (MICU1) and mitochondrial ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic β-cells. J. Biol. Chem. 287: 34445-34454.
De Marchi, U., J. Santo-Domingo, C. Castelbou, I. Sekler, A. Wiederkehr, and N. Demaurex. (2014). NCLX protein, but not LETM1, mediates mitochondrial Ca²⁺ extrusion, thereby limiting Ca²⁺-induced NAD(P)H production and modulating matrix redox state. J. Biol. Chem. 289: 20377-20385.
Dos Santos, G.R.R., A.C. Rezende Leite, N. Lander, M.A. Chiurillo, A.E. Vercesi, and R. Docampo. (2021). Trypanosoma cruzi Letm1 is involved in mitochondrial Ca transport, and is essential for replication, differentiation, and host cell invasion. FASEB J. 35: e21685.
Goetzl, E.J., V.H. Srihari, M. Mustapic, D. Kapogiannis, and G.R. Heninger. (2022). Abnormal levels of mitochondrial Ca channel proteins in plasma neuron-derived extracellular vesicles of early schizophrenia. FASEB J. 36: e22466.
Gyimesi, G. and M.A. Hediger. (2020). Sequence Features of Mitochondrial Transporter Protein Families. Biomolecules 10:.
Gyimesi, G. and M.A. Hediger. (2022). Systematic in silico discovery of novel solute carrier-like proteins from proteomes. PLoS One 17: e0271062.
Jiang, D., L. Zhao, and D.E. Clapham. (2009). Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca²⁺/H⁺ antiporter. Science 326: 144-147.
Jiang, D., L. Zhao, C.B. Clish, and D.E. Clapham. (2013). Letm1, the mitochondrial Ca²⁺/H⁺ antiporter, is essential for normal glucose metabolism and alters brain function in Wolf-Hirschhorn syndrome. Proc. Natl. Acad. Sci. USA 110: E2249-2254.
Kaiyrzhanov, R., S.E.M. Mohammed, R. Maroofian, R.A. Husain, A. Catania, A. Torraco, A. Alahmad, M. Dutra-Clarke, S. Grønborg, A. Sudarsanam, J. Vogt, F. Arrigoni, J. Baptista, S. Haider, R.G. Feichtinger, P. Bernardi, A. Zulian, M. Gusic, S. Efthymiou, R. Bai, F. Bibi, A. Horga, J.A. Martinez-Agosto, A. Lam, A. Manole, D.P. Rodriguez, R. Durigon, A. Pyle, B. Albash, C. Dionisi-Vici, D. Murphy, D. Martinelli, E. Bugiardini, K. Allis, C. Lamperti, S. Reipert, L. Risom, L. Laugwitz, M. Di Nottia, R. McFarland, L. Vilarinho, M. Hanna, H. Prokisch, J.A. Mayr, E.S. Bertini, D. Ghezzi, E. Østergaard, S.B. Wortmann, R. Carrozzo, T.B. Haack, R.W. Taylor, A. Spinazzola, K. Nowikovsky, and H. Houlden. (2022). Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement. Am J Hum Genet 109: 1692-1712.
Ko, J., Y.H. Lee, S.Y. Hwang, Y.S. Lee, S.M. Shin, J.H. Hwang, J. Kim, Y.W. Kim, S.W. Jang, Z.Y. Ryoo, I.K. Kim, S.E. Namkoong, and J.W. Kim. (2003). Identification and differential expression of novel human cervical cancer oncogene HCCR-2 in human cancers and its involvement in p53 stabilization. Oncogene 22: 4679-4689.
Li, Y., Q. Tran, R. Shrestha, L. Piao, S. Park, J. Park, and J. Park. (2019). LETM1 is required for mitochondrial homeostasis and cellular viability (Review). Mol Med Rep 19: 3367-3375.
Lim, S.G., K. Suk, and W.H. Lee. (2020). LETMD1 Regulates Phagocytosis and Inflammatory Responses to Lipopolysaccharide via Reactive Oxygen Species Generation and NF-κB Activation in Macrophages. J Immunol 204: 1299-1309.
Lin, Q.T. and P.B. Stathopulos. (2019). Molecular Mechanisms of Leucine Zipper EF-Hand Containing Transmembrane Protein-1 Function in Health and Disease. Int J Mol Sci 20:.
Nowikovsky, K., E.M. Froschauer, G. Zsurka, J. Samaj, S. Reipert, M. Kolisek, G. Wiesenberger, and R.J. Schweyen. (2004). The LETM1/YOL027 gene family encodes a factor of the mitochondrial K⁺ homeostasis with a potential role in the Wolf-Hirschhorn syndrome. J. Biol. Chem. 279: 30307-30315.
Santo-Domingo J. and Demaurex N. (2010). Calcium uptake mechanisms of mitochondria. Biochim Biophys Acta. 1797(6-7):907-12.
Shao, J., Z. Fu, Y. Ji, X. Guan, S. Guo, Z. Ding, X. Yang, Y. Cong, and Y. Shen. (2016). Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) forms a Ca²⁺/H⁺ antiporter. Sci Rep 6: 34174.
Tsai MF., Jiang D., Zhao L., Clapham D. and Miller C. (2014). Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1. J Gen Physiol. 143(1):67-73.
Waldeck-Weiermair, M., C. Jean-Quartier, R. Rost, M.J. Khan, N. Vishnu, A.I. Bondarenko, H. Imamura, R. Malli, and W.F. Graier. (2011). Leucine zipper EF hand-containing transmembrane protein 1 (Letm1) and uncoupling proteins 2 and 3 (UCP2/3) contribute to two distinct mitochondrial Ca²⁺ uptake pathways. J. Biol. Chem. 286: 28444-28455.
Wunderlich, J. (2022). Updated List of Transport Proteins in. Front Cell Infect Microbiol 12: 926541.
Zhang, X., G. Chen, Y. Lu, J. Liu, M. Fang, J. Luo, Q. Cao, and X. Wang. (2014). Association of mitochondrial letm1 with epileptic seizures. Cereb Cortex 24: 2533-2540.
Examples:
TC#	Name	Organismal Type	Example
2.A.97.1.1	The leucine zipper-EF band transmembrane protein 1, LetM1. Reduces mitochondrial Ca²⁺ uptake in response to cytoplasmic Ca²⁺entry in pancreatic beta cells (Alam et al. 2012). LetM1 is a cation/Ca²⁺ transporter, with apparent affinities of cations in the order Ca²⁺ = Mn²⁺ > Gd³⁺ = La³⁺ > Sr²⁺ >> Ba²⁺, Mg²⁺, K⁺, Na⁺(Shao et al. 2016). The LetM1 turnover rate is only 2 Ca²⁺/sec with a K_m of ~ 25 microM. LetM1 mediates electroneutral 1 Ca²⁺/2 H⁺ antiport (Tsai et al. 2013). However, NCLX (2.A.19.4.4), rather than LetM1, may mediate miltochondrial Ca²⁺ extrusion (De Marchi et al. 2014). Letm1 is associated with seizure attacks in Wolf-Hirschhorn syndrome, and its inhibition and mitochondrial dysfunctions contribute to the development of epileptic seizures. An appropriate LetM1 level may be critical for maintaining normal neuronal functions (Zhang et al. 2014). Glu221 in the mouse orthologue is essential. The protein is a hexamer with a central cavity and exhibits two different conformational states (Shao et al. 2016). Abnormal levels occur in plasma neuron-derived extracellular vesicles in early schizophrenia and other neurodevelopmental diseases (Goetzl et al. 2022). LetM1 has an osmoregulatory function controlling mitochondrial volume and ion homeostasis (Kaiyrzhanov et al. 2022). Bi-allelic LETM1 variants are associated with defective mitochondrial K⁺ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies (Kaiyrzhanov et al. 2022).	Animals	*LetM1 of Homo sapiens* (O95202)**

2.A.97.1.2	Mitochondrial distribution and morphology protein Mdm38p (Yol027c)	Yeast	*Mdm38p of Saccharomyces cerevisiae* (Q08179)**

2.A.97.1.3	*The mitochondrial LetM1 Ca²⁺/H⁺ antiporter (Jiang et al., 2009).*	Metazoa	*LetM1 of Drosophila melanogaster* (P91927)**

2.A.97.1.4	*The yeast Mdm38 (LETM1; YLH47) is a homologue of the human LETM1, the candidate gene for seizures in Wolf-Hirchhorn syndrome. They may be K⁺/H⁺ antiporters (Zotova et al., 2010).*	Yeast	*LETM1 of Saccharomyces cerevisiae* (Q06493)**

2.A.97.1.5	Letm1 RBD domain-containing protein of 479 aas and 1 or 2 TMSs. Trypanosoma cruzi Letm1 is involved in mitochondrial Ca²⁺ transport, and is essential for replication, differentiation, and host cell invasion (Dos Santos et al. 2021).		LetM1 of Trypanosoma cruzi

2.A.97.1.6	Mitochondrial LETM2 or SLC55A2 of 491 aas and probably 2 TMSs at approximately residues 130 and 180 (Gyimesi and Hediger 2022).		LETM2 of Homo sapiens

2.A.97.1.7	LetM1 domain-containing protein, LetD1, LTMD1, SLC55A3, of 360 aas and 2 central TMSs (at residues 90 and 140) as well as two possible TMSs at the N- and C-termini of the protein. It is involved in tumorigenesis and may function as a negative regulator of the p53/TP53 pathway (Ko et al. 2003). It may play an essential role for mitochondrial structure and function, and thermogenesis of brown adipocytes. It may also regulate phagocytosis and inflammatory responses to lipopolysaccharide in macrophages (Lim et al. 2020).		LetD1 of Homo sapiens

2.A.97.1.8	LetM1-like protein of 802 aas and 2 TMSs. [There are two mildly hydrophobic potential TMSs preceding each of the two strongly hydrophobic TMSs (residue #s 290 - 400 and 470 - 530]. LetM1 exports Ca²⁺ and K⁺ and imports H⁺ (Wunderlich 2022).		LetM1-like protein of Plasmodium falciparum