SLC9B2 (NHA2/NHEDC2) in Male Fertility

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1		Paul F. James	--	1149	2023-10-25 20:24:56	\|
2	layout	Jessie Wu	-2 word(s)	1147	2023-10-26 02:47:48	\|

SLC9B2 (NHA2/NHEDC2) in Male Fertility

This entry is adapted from the peer-reviewed paper 10.3390/ijms241914981

The SLC9B2 gene encodes the NHA2 protein (also known as NHEDC2). While NHA2 has been shown to be important for regulating various aspects of physiology such as blood pressure, this protein has also been implicated as being important for male fertility in mice. What is known about NHA2 and its potentially important role in male fertility is emphasized.

β-cells NHA2 NHA1 cKO

1. Molecular Genetics and Expression Patterns

SLC9B2 (NHA2/NHEDC2) is located in tandem with SLC9B1 on human Chromosome 4 and is comprised of 13 exons. In contrast to SLC9B1, SLC9B2 exhibits near ubiquitous expression, including expression in testis and sperm, in both mouse and human. In mature mouse sperm, NHA2 has been reported to reside in the principal piece ^[1].

NHA2 subcellular location seems to be widespread as it has been reported to localize to multiple cellular compartments in various cells. In mouse pancreatic β-cells, NHA2 localizes to endosomes and synaptic-like microvesicles and, similarly, was found to localize to late endosomes and lysosomes in mouse osteoclasts ^[2]^[3]. NHA2 is also reported to localize to mitochondria in both mouse osteoclasts and rat kidney cells ^[4]^[5]. Additionally, NHA2 was reported to localize to the plasma membrane of mouse kidney cells, mouse osteoclasts, and MDCK cells (canine kidney cells) ^[3]^[6]^[7]^[8].

Physiologically, NHA2 has been shown to be important for osteoclast function, insulin secretion, and nephron function where it has been implicated in playing a significant role in maintaining salt balance to support normotension ^[2]^[3]^[6]^[9]. NHA2 was reported to localize to the distal convoluted tubules of mouse kidney cells, and subcellularly, was found to localize to endosomes in mpkDCT4 cells, a mouse distal convoluted tubule epithelial cell line, although NHA2 was found to not influence endosomal pH homeostasis ^[9]. NHA2 knockout mice display a Gitelman syndrome-like phenotype, characterized by reduced blood pressure, normocalcemic hypocalciuria, and increased urinary aldosterone excretion. Mechanistically, NHA2 appears to regulate blood pressure through a WNK4-NCC pathway in the mouse kidney ^[9]. For a review on NHA2 work from the Reymond, von Ballmoos, and Fuster groups, see ^[1].

2. Sperm Physiology and Fertility

As with NHA1, two lines of NHA2 KO mice have been generated. The Liu group generated conditional NHA2 knockout mice in the same manner as the NHA1 conditional knockout mice ^[10] such that a conditional global knockout of NHA2 occurred. The NHA2 conditional knockout (NHA2 cKO) mice exhibited a very similar phenotype to the NHA1 cKO mice in that they were subfertile due to reduced sperm motility. Similar to the NHA1 cKO mice, there are no grossly abnormal testis or sperm morphologies and the sperm have reduced cAMP levels and sAC protein levels. Unfortunately, there is no information available regarding whether cAMP analogs can restore motility and fertility of NHA2 cKO sperm. Of interest, the NHA1 cKO mouse testis had significantly upregulated NHA2 mRNA and the NHA2 cKO mouse testis had significantly upregulated NHA1 mRNA, suggesting a functional redundancy for the two transporters ^[10]. Interestingly, there are no reports of a subfertile male phenotype from the second group that generated NHA2 knockout mice ^[2]^[9].

NHA1/NHA2 double knockout mice (NHA1/2 dKO) were reported to have been generated ^[10] however, as noted by ^[11] the likelihood of generating these NHA1/2 dKO by cross-breeding the NHA1 cKO and NHA2 cKO mice is low because Slc9B1 and Slc9B2 are located in tandem on chromosome 3 in mice. These two genes are separated by less than 18,500 bp on mouse chromosome 3 (according to the chromosomal locations for these two genes provided in ^[12]), making the occurrence of a crossover event happening that would result in positioning both of the individual knockout alleles on the same chromosome extremely rare. Therefore, it would be expected to take a cumbersomely large number of matings between the single KO mice to generate the NHA1/2 dKO founder and thus the actual genotypes of the NHA1/2 dKO animals used in these studies are unclear. In any case, the NHA1/2 dKO sperm were reported to have almost no motility resulting in completely infertile males. Likely because the NHA1/2 dKO mouse sperm were reported to have significantly reduced cAMP and sAC protein levels ^[10]. Similar to the NHA2 cKO sperm, there is no information about the ability of cAMP analogs to restore motility to the NHA1/2 dKO sperm.

3. NHA2 Transport Activity

Initial attempts to characterize the transport activity of the human NHA2 protein found that heterologous expressed NHA2 in salt-sensitive yeast strains was able to transport either Na⁺ or Li⁺, consistent with conventional Na⁺/H⁺ exchanger activity ^[4]^[13]. Later characterization of human NHA2 was accomplished by overexpressing green fluorescent protein (GFP)-tagged human NHA2 fusion protein in MDCK cells and it was found that NHA2 acts to export Li⁺ out of the cell in exchange for the import of Na⁺, however, in the presence of a high extracellular proton concentration (acidic conditions outside the cell) NHA2 could mediate Li⁺ efflux in exchange for H⁺ import. This NHA2 activity was inhibited by cytosolic acidification as no Na⁺ influx was seen after the cells were acid loaded ^[7]. In addition, there appears to be a consensus that NHA2 does not transport K⁺ and that its activity is not electrogenic ^[4]^[7]^[14]. Furthermore, NHA2 activity is resistant to amiloride but sensitive to the sodium–glucose transporter inhibitor phloretin ^[6]^[7]. Interestingly, exogenously expressed NHA2 coimmunoprecipitated with the V-type H⁺-ATPase in a kidney cell line and the V-type H⁺-ATPases inhibitor bafilomycin was able to block NHA2-mediated Li⁺ tolerance ^[7]. These findings led to a model proposing that NHA2 cation extrusion is driven by the proton gradient setup by the H⁺ efflux driven by the V-type H⁺-ATPase ^[7], therefore suggesting that human NHA2 is a H⁺-driven cation (Na⁺/Li⁺) exporter with activity similar to the prokaryotic NHAs. It is currently unclear whether NHA2 and V-Type H⁺-ATPases are also functionally coupled in spermatozoa; future experiments are needed to examine whether these proteins share similar transport characteristics in sperm and kidney cells.

More recent experiments found that human NHA2 preferentially forms stable homodimers in membranes ^[15] and exhibits electroneutral NHE activity when in proteoliposomes ^[14]. In addition, the structure of the bison NHA2 protein was recently solved and found to possess 14 transmembrane domains and form a homodimer ^[16]. Solid-state membrane-based electrophysiology experiments on proteoliposome-reconstituted bison NHA2 revealed that bison NHA2 mediates Na⁺/H⁺ exchange (can also transport Li⁺/H⁺) but in a pH dependent manner, specifically Na⁺(Li⁺)/H⁺ exchange was severely inhibited by a low pH outside the liposome relative to inside the liposomes. All together, these findings suggest that NHA2 operates as an electroneutral exchanger to drive Na⁺ efflux ^[16]. However, biochemical analyses of NHA2 in its native environment is lacking and whether or not NHA2 plays a role in organellar pH homeostasis such as endosomal pH regulation needs to be clarified. Additionally, how NHA2 transport activity influences sperm physiology remains unknown.

4. NHA2 and Human Fertility

A recent study detected NHA2 protein in human sperm via mass spectroscopy ^[16], however its exact contribution to human male fertility is still unknown.

References

Chen, S.R.; Chen, M.; Deng, S.L.; Hao, X.X.; Wang, X.X.; Liu, Y.X. Sodium–Hydrogen Exchanger NHA1 and NHA2 Control Sperm Motility and Male Fertility. Cell Death Dis. 2016, 7, e2152–e2159.
Hofstetter, W.; Siegrist, M.; Simonin, A.; Bonny, O.; Fuster, D.G. Sodium/Hydrogen Exchanger NHA2 in Osteoclasts: Subcellular Localization and Role in Vitro and in Vivo. Bone 2010, 47, 331–340.
Fuster, D.G.; Zhang, J.; Shi, M.; Alexandru Bobulescu, I.; Andersson, S.; Moe, O.W. Characterization of the Sodium/Hydrogen Exchanger NHA2. J. Am. Soc. Nephrol. 2008, 19, 1547–1556.
Battaglino, R.A.; Pham, L.; Morse, L.R.; Vokes, M.; Sharma, A.; Odgren, P.R.; Yang, M.; Sasaki, H.; Stashenko, P. NHA-Oc/NHA2: A Mitochondrial Cation-Proton Antiporter Selectively Expressed in Osteoclasts. Bone 2008, 42, 180–192.
Xiang, M.; Feng, M.; Muend, S.; Rao, R. A Human Na+/H+ Antiporter Sharing Evolutionary Origins with Bacterial NhaA May Be a Candidate Gene for Essential Hypertension. Proc. Natl. Acad. Sci. USA 2007, 104, 18677–18681.
Kondapalli, K.C.; Kallay, L.M.; Muszelik, M.; Rao, R. Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter, to a Plasma Membrane H+ Gradient. J. Biol. Chem. 2012, 287, 36239–36250.
Kondapalli, K.C.; Todd Alexander, R.; Pluznick, J.L.; Rao, R. NHA2 Is Expressed in Distal Nephron and Regulated by Dietary Sodium. J. Physiol. Biochem. 2017, 73, 199–205.
Anderegg, M.A.; Albano, G.; Hanke, D.; Deisl, C.; Uehlinger, D.E.; Brandt, S.; Bhardwaj, R.; Hediger, M.A.; Fuster, D.G. The Sodium/Proton Exchanger NHA2 Regulates Blood Pressure through a WNK4-NCC Dependent Pathway in the Kidney. Kidney Int. 2021, 99, 350–363.
Ho, T.M.; Berger, S.; Müller, P.; Simonin, C.; Reymond, J.; Von Ballmoos, C.; Fuster, D.G. Physiological and Molecular Function of the Sodium/Hydrogen Exchanger NHA2 (SLC9B2). Chimia 2022, 76, 1019.
Anderegg, M.A.; Gyimesi, G.; Ho, T.M.; Hediger, M.A.; Fuster, D.G. The Less Well-Known Little Brothers: The SLC9B/NHA Sodium Proton Exchanger Subfamily—Structure, Function, Regulation and Potential Drug-Target Approaches. Front. Physiol. 2022, 13, 898508.
Holmes, R.S.; Spradling-Reeves, K.D.; Cox, L.A. Evolution of Vertebrate Solute Carrier Family 9B Genes and Proteins (SLC9B): Evidence for a Marsupial Origin for Testis Specific SLC9B1 from an Ancestral Vertebrate SLC9B2 Gene. J. Phylogenet. Evol. Biol. 2016, 4, 167.
Huang, X.; Morse, L.R.; Xu, Y.; Zahradka, J.; Sychrová, H.; Stashenko, P.; Fan, F.; Battaglino, R.A. Mutational Analysis of NHAoc/NHA2 in Saccharomyces Cerevisiae. Biochim. Biophys. Acta-Gen. Subj. 2010, 1800, 1241–1247.
Uzdavinys, P.; Coinçon, M.; Nji, E.; Ndi, M.; Winkelmann, I.; Von Ballmoos, C.; Drew, D. Dissecting the Proton Transport Pathway in Electrogenic Na+/H+ Antiporters. Proc. Natl. Acad. Sci. USA 2017, 114, E1101–E1110.
Landreh, M.; Marklund, E.G.; Uzdavinys, P.; Degiacomi, M.T.; Coincon, M.; Gault, J.; Gupta, K.; Liko, I.; Benesch, J.L.P.; Drew, D.; et al. Integrating Mass Spectrometry with MD Simulations Reveals the Role of Lipids in Na+/H+ Antiporters. Nat. Commun. 2017, 8, 13993.
Matsuoka, R.; Fudim, R.; Jung, S.; Zhang, C.; Bazzone, A.; Chatzikyriakidou, Y.; Robinson, C.V.; Nomura, N.; Iwata, S.; Landreh, M.; et al. Structure, Mechanism and Lipid-Mediated Remodeling of the Mammalian Na+/H+ Exchanger NHA2. Nat. Struct. Mol. Biol. 2022, 29, 108–120.
Grahn, E.; Kaufmann, S.V.; Askarova, M.; Ninov, M.; Welp, L.M.; Berger, T.K.; Urlaub, H.; Kaupp, U.B. Control of Intracellular PH and Bicarbonate by CO2 Diffusion into Human Sperm. Nat. Commun. 2023, 14, 5395.

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Update Date: 26 Oct 2023

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