Physiological and Molecular Function of the Sodium/Hydrogen Exchanger NHA2 (SLC9B2).

NHA2, also known as SLC9B2, is an orphan intracellular Na+/H+ exchanger (NHE) that has been associated with arterial hypertension and diabetes mellitus in humans. The objective of this NCCR TransCure project was to define the physiological and molecular function of NHA2, to develop a high resolution kinetic transport assay for NHA2 and to identify specific and potent compounds targeting NHA2. In this review, we summarize the results of this highly interdisciplinary and interfaculty effort, led by the groups of Proffs. Jean-Louis Reymond, Christoph von Ballmoos and Daniel Fuster.

The sodium/proton exchanger NHA2 regulates blood pressure through a WNK4-NCC dependent pathway in the kidney.

NHA2 is a sodium/proton exchanger associated with arterial hypertension in humans, but the role of NHA2 in kidney function and blood pressure homeostasis is currently unknown. Here we show that NHA2 localizes almost exclusively to distal convoluted tubules in the kidney. NHA2 knock-out mice displayed reduced blood pressure, normocalcemic hypocalciuria and an attenuated response to the thiazide diuretic hydrochlorothiazide. Phosphorylation of the thiazide-sensitive sodium/chloride cotransporter NCC and its upstream activating kinase Ste20/SPS1-related proline/alanine rich kinase (SPAK), as well as the abundance of with no lysine kinase 4 (WNK4), were significantly reduced in the kidneys of NHA2 knock-out mice. In vitro experiments recapitulated these findings and revealed increased WNK4 ubiquitylation and enhanced proteasomal WNK4 degradation upon loss of NHA2. The effect of NHA2 on WNK4 stability was dependent from the ubiquitylation pathway protein Kelch-like 3 (KLHL3). More specifically, loss of NHA2 selectively attenuated KLHL3 phosphorylation and blunted protein kinase A- and protein kinase C-mediated decrease of WNK4 degradation. Phenotype analysis of NHA2/NCC double knock-out mice supported the notion that NHA2 affects blood pressure homeostasis by a kidney-specific and NCC-dependent mechanism. Thus, our data show that NHA2 as a critical component of the WNK4-NCC pathway and is a novel regulator of blood pressure homeostasis in the kidney.

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+ /H+ antiporter NHA2.

The human Na+ /H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50-70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity.

The Less Well-Known Little Brothers: The SLC9B/NHA Sodium Proton Exchanger Subfamily-Structure, Function, Regulation and Potential Drug-Target Approaches.

The SLC9 gene family encodes Na+/H+ exchangers (NHEs), a group of membrane transport proteins critically involved in the regulation of cytoplasmic and organellar pH, cell volume, as well as systemic acid-base and volume homeostasis. NHEs of the SLC9A subfamily (NHE 1-9) are well-known for their roles in human physiology and disease. Much less is known about the two members of the SLC9B subfamily, NHA1 and NHA2, which share higher similarity to prokaryotic NHEs than the SLC9A paralogs. NHA2 (also known as SLC9B2) is ubiquitously expressed and has recently been shown to participate in renal blood pressure and electrolyte regulation, insulin secretion and systemic glucose homeostasis. In addition, NHA2 has been proposed to contribute to the pathogenesis of polycystic kidney disease, the most common inherited kidney disease in humans. NHA1 (also known as SLC9B1) is mainly expressed in testis and is important for sperm motility and thus male fertility, but has not been associated with human disease thus far. In this review, we present a summary of the structure, function and regulation of expression of the SLC9B subfamily members, focusing primarily on the better-studied SLC9B paralog, NHA2. Furthermore, we will review the potential of the SLC9B subfamily as drug targets.

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.

SLC9B genes and proteins are members of the sodium/lithium hydrogen antiporter family which function as solute exchangers within cellular membranes of mammalian tissues. SLC9B2 and SLC9B1 amino acid sequences and structures and SLC9B-like gene locations were examined using bioinformatic data from several vertebrate genome projects. Vertebrate SLC9B2 sequences shared 56-98% identity as compared with ∼50% identities with mammalian SLC9B1 sequences. Sequence alignments, key amino acid residues and conserved predicted transmembrane structures were also studied. Mammalian SLC9B2 and SLC9B1 genes usually contained 11 or 12 coding exons with differential tissue expression patterns: SLC9B2, broad tissue distribution; and SLC9B1, being testis specific. Transcription factor binding sites and CpG islands within the human SLC9B2 and SLC9B1 gene promoters were identified. Phylogenetic analyses suggested that SLC9B1 originated in an ancestral marsupial genome from a SLC9B2 gene duplication event.