Molecular cloning and functional expression of a rat Na+/H+ exchanger (NHE5) highly expressed in brain

We report here the cloning, primary structure, heterologous expression, tis sue distribution, and localization of a cDNA encoding rat NHE5, a fifth mem ber of the mammalian plasma membrane Na+/H+ exchanger (NHE) gene family. Th e full-length open reading frame as well as 34 nucleotides of 5'-untranslat ed and 1443 nucleotides of 3'-untranslated sequences were obtained using a polymerase chain reaction strategy involving reverse transcription-polymera se chain reaction and 5'/3'-rapid amplification of cDNA ends. The NHE5 cDNA encodes a protein of 898 amino acids with a calculated M-r of 99,044 and i s predicted to contain 11-13 transmembrane domains. An amino acid compariso n of the coding region of rat NHE5 reveals 95% identity with human NHE5. No rthern hybridization analysis showed that high level expression of NHE5 mRN A is restricted to brain. Transfection of the coding region of rat NHE5 int o NHE-deficient PS120 cells resulted in Na+/H+ exchange activity that was r elatively insensitive to the amiloride analogue, 5-(N-ethyl-N-isopropyl) am iloride, with a half-maximal inhibitory concentration (IC50) of 1.53 +/- 0. 25 mu M. In situ hybridization of rat brain sections revealed significant N HE5 mRNA levels in the dentate gyrus with lower levels observed in the hipp ocampus and cerebral cortex. These results suggest a specialized role for t his fifth NHE isoform in neuronal tissues.