Mutational Analysis The Function Of NHA2 In Yeast And The Functional Study Of It's Expression And Roles In The Mammalian Cell And Mouse

NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc in eukaryotes.The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of HsNHA2 above all. The growth phenotypes of cells expressing HsNHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth. We also use RNAKL to induce the RAW264.7 differentiated into osteoclast-like cells. At different growth stage after RNAKL induction, we detect the NHA2 expression respectively. At last, we use NHA2 knock out mice to detect the bone mineral density and the activity that bone marrow cell differentiated into mature osteoclast.Mutations in aspartic acid residues 278 and 279 completely abolished the ability of BW31a cells grow into NaCl and each residue is essential for activity. Yeast expressing the double mutant F357 F437 can not grow into 0.4 M NaCl, suggesting that these residues are essential for antiporter activity. HsNHA2 can form a dimer since simultaneous expression of D278C and D279C mutants partially rescues the salt-sensitive growth phenotype. Both deletion mutants (G2-V187 and S107-L115) fail to rescue the ability of BW31a cells to grow in 0.2 M NaCl, indicating that both regions are required for activity of HsNHA2.During steoclast differentiation, we found that the NHA2 expression gradually increase in vitro. It can be detected after inducing 2 days by RANKL, and attain to peak in 5 days, which indicate that NHA2 mainly express in the late stage of osteoclast differentiation.Evolutionarily conserved amino acids are required for full antiporter function. Mutations in these amino acid residues may impact HsNHA2 activity and therefore osteoclast function in vitro and in vivo. Our findings lead to a model for transport mechanism that was compared to the well-known electroneutral NHE and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.