Na+/H+ exchangers of the NHE superfamily mediate the transmembrane exchange of cations with protons to regulate salt, pH and water homeostasis. We have uncovered an evolutionarily ancient subgroup of endosomal NHE that includes yeast Nhx1 and mammalian NHE6, 7 and 9. In yeast, Nhx1 localizes to the late endosome where it regulates luminal pH to control vesicle trafficking and delivery of the multivesicular body (MVB) to the vacuole for degradation. In mammals, the MVB pathway is important in HIV biogenesis, drug detoxification, erythrocyte maturation, and protein degradation. Defects in this pathway are likely to lead to lysosomal storage disorders and concomitant neurological and kidney dysfunction. Inhibitors of endosomal NHE offer a therapeutic potential to offset defects in endosome acidification seen in Dent's and Fanconi disease, and as antiviral agents. The goal of this proposal is to extend our understanding of yeast Nhx1 function and extrapolate our findings to mammalian cells.
In Aim 1, we will use a combination of yeast genetics, biochemical assays of trafficking, and electron microscopy to define the precise pH-dependent step in lysosomal biogenesis. In parallel, we will test the hypothesis that NHE6 and/or NHE9 localize and function in MVB bodies in a mammalian cell culture model.
In Aim 2, we will evaluate synthetic variants of exoporide, a novel amiloride analog, to find a selective inhibitor of intracellular NHE. An immediate goal of this proposal is to complete ongoing studies that seek to derive a global view of the role of cation/proton exchange by Nhx1 (Aim 3). To this end, we will continue our analysis of the genetic basis for pH regulation (pHome) and identify genes and cellular pathways that interact with Nhx1 (phenome).
In Aim 4, we will assess an emerging homology model of NHE, based on the crystal structure of E. coli NhaA, using structure- bioinformatics driven mutagenesis in conjunction with phenotype screening in yeast. These studies will focus on defining the molecular basis for differences in ion selectivity and inhibitor sensitivity between the intracellular and plasma membrane subtypes of NHE, provide insight into the mechanism of transport by the NHE superfamily, and serve as a template for the design of novel NHE inhibitors. In summary, we propose a multidisciplinary approach that targets the function and mechanism of a clinically and physiologically important family of membrane transport proteins.

Public Health Relevance

This proposal targets a newly discovered but evolutionarily ancient family of ion transporters that regulate the movement of salt, water and acid equivalents across the boundaries and compartments of all cells. We plan to define the function of these proteins using parallel approaches in yeast and cultured mammalian cells, and identify new drugs using a novel screening strategy. These drugs may offer therapeutic benefits in kidney storage diseases (Dent's and Fanconi), and against envelope viruses such as HIV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054214-12
Application #
8091274
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Mullins, Christopher V
Project Start
2008-06-01
Project End
2012-05-31
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
12
Fiscal Year
2011
Total Cost
$307,407
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Kondapalli, Kalyan C; Llongueras, Jose P; Capilla-González, Vivian et al. (2015) A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma. Nat Commun 6:6289
Prasad, Hari; Rao, Rajini (2015) The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease. J Biol Chem 290:5311-27
Kondapalli, Kalyan C; Hack, Anniesha; Schushan, Maya et al. (2013) Functional evaluation of autism-associated mutations in NHE9. Nat Commun 4:2510
Kondapalli, Kalyan C; Kallay, Laura M; Muszelik, Melanie et al. (2012) Unconventional chemiosmotic coupling of NHA2, a mammalian Na+/H+ antiporter, to a plasma membrane H+ gradient. J Biol Chem 287:36239-50
Chanroj, Salil; Lu, Yongxian; Padmanaban, Senthilkumar et al. (2011) Plant-specific cation/H+ exchanger 17 and its homologs are endomembrane K+ transporters with roles in protein sorting. J Biol Chem 286:33931-41
Brett, Christopher L; Kallay, Laura; Hua, Zhaolin et al. (2011) Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae. PLoS One 6:e17619
Kallay, Laura M; Brett, Christopher L; Tukaye, Deepali N et al. (2011) Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation. J Biol Chem 286:44067-77
Schushan, Maya; Xiang, Minghui; Bogomiakov, Pavel et al. (2010) Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension. J Mol Biol 396:1181-96
Xiang, Minghui; Feng, Mingye; Muend, Sabina et al. (2007) A human Na+/H+ antiporter sharing evolutionary origins with bacterial NhaA may be a candidate gene for essential hypertension. Proc Natl Acad Sci U S A 104:18677-81
Hill, Jennifer K; Brett, Christopher L; Chyou, Anthony et al. (2006) Vestibular hair bundles control pH with (Na+, K+)/H+ exchangers NHE6 and NHE9. J Neurosci 26:9944-55

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