Characterization of the sodium/hydrogen exchanger NHA2

Abstract

Cation/proton exchange has been recognized for decades in mammalian mitochondria, but the exchanger proteins have eluded identification. In this study, a cDNA from a human brain library, previously designated NHA2 in the genome, was cloned and characterized. The NHA2 transcript bears more similarity to prokaryotic than known eukaryotic sodium/proton exchangers, but it was found to be expressed in multiple mammalian organs and cultured cells. A mAb to NHA2 was generated and found to label an approximately 55-kD native protein in multiple tissues and cell lines. The specificity of this antibody was confirmed by demonstrating the loss of the native NHA2 band on immunoblots when cultured cells were treated with NHA2-specific small interfering RNA. Although NHA2 protein was detected in multiple organs, within each, its expression was restricted to specific cell types. In the kidney, co-localization with calbindin 28k and reverse transcription-PCR of microdissected tubules revealed that NHA2 is limited to the distal convoluted tubule. In cell lines, native NHA2 was localized both to the plasma membrane and to the intracellular compartment; immunogold electron microscopy of rat distal convoluted tubule demonstrated NHA2 predominantly but not exclusively on the inner mitochondrial membrane. Furthermore, co-sedimentation of NHA2 antigen and mitochondrial membranes was observed with differential centrifugation, and two mitochondrial markers co-localized with NHA2 in cultured cells. Regarding function, human NHA2 reversed the sodium/hydrogen exchanger-null phenotype when expressed in sodium/hydrogen exchanger-deficient yeast and restored the ability to defend high salinity in the presence of acidic extracellular pH. In summary, NHA2 is a ubiquitous mammalian sodium proton/exchanger that is restricted to the distal convoluted tubule in the kidney.

Figure 1.

Sequence comparison of NHA2. Amino acid alignment of human (Hs), mouse (Mm), and rat (Rn) NHA2. Sequence identity is 81% between human and mouse or human and rat NHA2. Epitope of MAB45D12D is marked in yellow highlight.

Figure 2.

NHA2 mRNA expression in cells and tissue. (A) RNA blot of three human cell lines using a ³²P-labeled human NHA2 cDNA probe. Ten micrograms of total RNA was loaded per lane. Human NHA2 transcript size is approximately 2.4 kb. (B) RT-PCR of mouse NHA2 from various tissues. One microgram of total RNA was subjected to oligo-dT–primed reverse transcription followed by PCR using mouse NHA2- or β-actin–specific primers for 35 cycles.

Figure 3.

Immunoblot of native and overexpressed human NHA2. (A) NRK cells were transfected with two different concentrations of a mix of two rat NHA2-specific siRNA (middle lane 50 pg, right lane 30 pg) and compared with mock-transfected cells. Twenty-four hours after transfection, 50 μg of total lysate protein was separated by SDS-PAGE and endogenous NHA2, NHE1, or NHE8 protein levels were detected by immunoblotting using MAB45D12D, monoclonal anti-NHE1, or anti-NHE8 antibodies. (B) MAB45D12D does not react with NHA1. Human C-terminally GFP-tagged NHA1 or empty vector was transiently expressed in AP-1 cells. Fifty micrograms of total protein was separated by SDS-PAGE and blotted with MAB45D12D or monoclonal anti-GFP antibody. (C) Expression of NHA2 in various mouse tissues. Fifty micrograms of total lysate protein were separated by SDS-PAGE followed by immunoblotting with MAB45D12D. (D) Surface expression of native and overexpressed NHA2 in different cells assessed by surface biotinylation. Native NHA2 is present on the surface membrane of RBC (a), a rat pancreatic β cell line (d), and HEK293 cells transiently transfected with human C-terminally HA-tagged NHA2 (c), but native NHA2 is absent from the surface membrane in HEK293 cells (b). Endogenous NHE1 was used as biotinylation control (e).

Figure 4.

Tissue distribution of NHA2 in the mouse assessed by immunohistochemistry. Tissue was harvested and prepared as described in the Concise Methods section. MAB45D12D was used for staining. Tissues are as follows: Kidney cortex (A), heart muscle (B), lung (C), brain cortex (D), jejunum (E), and mammary gland (F).

Figure 5.

Intracellular localization of endogenous NHA2. (A) Fluorescence immunocytochemistry. Bar = 10 μm. NRK cells were treated with the specific markers (mitochondria: Mitotracker [B] and α-CPS1 [E]; Golgi: α-GOLPH4 [H]; ER: α-PDI [K]) and then with MAB45D12D (α-NHA2 [A, D, G, and J]). Merged images (C, F, I, and L) show overlap of the organellar markers with NHA2. Note the absence of plasmalemmal staining for NHA2. (B) Mitochondrial isolation of HEK293 and NRK cells. Native NHA2 is present in the 3000 × g pellets as the mitochondrial proteins Tom40 and CPS1. LAMP1, a lysosomal membrane protein, partially co-purifies with the mitochondrial pellet but is also present in the 3000 × g supernatant. (C) Fluorescence immunocytochemical images of NRK cells treated with Mitotracker and stained with MAB45D12D. Disappearance of the MAB45D12D signal after siRNA transfection. (D and E) Immunogold electron microscopy of rat DCT cells using MAB45D12D. NHA2 is present on the inner mitochondrial membrane. Bar = 1 μm. (F) Quantitative gold particle density of rat DCT. Bars and error bars are means ± SD (n = 56). Background particle density (gold-conjugated secondary antibody only) was <0.5/μm².

Figure 6.

NHA2 localizes to DCT. (A) Immunofluorescence confocal microscopy of rat kidney cortex showing co-staining of calbindin 28k (red) and NHA2 (green) in distal tubules. (B) RT-PCR of NHA2 with appropriate control transcripts from microdissected rat tubules. Microdissection and RT-PCR were performed as described in the Concise Methods section. NHA2 could be amplified from DCT–cortical collecting duct (CCD) section. Note that separation of DCT from CCD was not possible because of technical limitations.

Figure 7.

Functional complementation of a salt-sensitive S. cerevisiae strain by heterologous expression of human NHA2. (A) Salt-sensitive yeast strain AXT3 was transformed with S. cerevisiae NHE (ScNha1 and ScNhx1), human NHA2, or empty vector p426TEF. Cells (5 × 10⁴) were plated with serial 10-fold dilutions (left to right) on AP medium plates with indicated Na⁺ concentrations and pH. Growth was assessed after 4 d. (B) S. cerevisiae AXT3 strain expressing C-terminally HA-tagged expression constructs. Equal amounts of yeast lysates from 10 OD units were loaded, separated by SDS-PAGE, and immunoblotted with a polyclonal anti-HA antibody.