Samir Nath

Structure/function studies of mammalian Na-H exchangers--an update.

Four mammalian Na+/H+ exchangers have recently been cloned. Despite the structural similarity, these Na+/H+ exchanger isoforms differ in kinetic characteristics and their response to external stimuli. The present review deals with the recent developments in their functional characterization and their short‐term regulation.

Molecular Properties, Kinetics And Regulation Of Mammalian Na+/H+ Exchangers

Na+/H+exchange was first described by Murer, Hopfer and Kinne [19] in renal brush border membrane vesicles. This process is mediated by Na+/H+exchangers which catalyze the exchange of extracellular Na+for intracellular H with a stoichiometry of 1:1. Na+/H+exchangers have multiple functions, including pH homeostasis, volume regulation, cell proliferation, and transcellular Na+absorption [reviewed in 12]. In no cell is it the only mechanism for any one of these functions. For instance, multiple mechanisms of pH homeostasis are present in most eukaryotic cells including a c┌/HCO3-exchanger, a NaHCO3co-transporter, a Na+- dependent cr/HCO3-exchanger and multiple mechanisms of hT extrusion [reviewed in 15], including the H-K-ATPase pump. In this review, we will focus on recent advances in identification and understanding of the structure/function relationships and acute protein kinase regulation of members of the mammalian Na+/H+exchanger gene family.

NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation.

The cloned epithelial cell-specific Na+/H+exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor (FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), and fetal bovine serum (FBS) through a change in maximal velocity of the transporter. In the present study, we used COOH-terminal truncation mutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at the truncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [a Ca2+/calmodulin (CaM) inhibitor] were studied. Truncation mutant E2/660, but not E2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGF stimulated E2/540 but not E2/499. The most truncated mutant, E2/499, was stimulated by FBS. W-13 stimulated the basal activity of the wild-type NHE2. However, W-13 had no effect on E2/755. By monitoring the emission spectra of dansylated CaM fluorescence, we showed that dansylated CaM bound directly to a purified fusion protein of glutathione S-transferase and the last 87 amino acids of NHE2 in a Ca2+-dependent manner, with a stoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized into separate stimulatory and inhibitory growth factor/protein kinase regulatory subdomains. This organization of growth factor/protein kinase regulatory subdomains is very similar to that of NHE3, suggesting that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar despite the minimal amino acid identity in this part of the two proteins.

Molecular Studies of Members of the Mammalian Na+/H+ Exchanger Gene Family

The brief history of the contribution of molecular biologic studies to the understanding of the Na+/H+ exchanger gene family is not unlike the history of studies of other transport proteins. Many years of results from physiologic and biochemical studies provided the background to allow strategies for the molecular recognition of an initial member of the Na+/H+ exchanger gene family. This was followed by recognition of the existence of a gene family, which even now is only partially defined. Rapid advances followed concerning location, regulation, and structure/function relationships, all of which have served to extend the previous physiologic studies. Current studies involve “torturing” the specific transport proteins by deletion and point mutation and creation of chimeric constructs to further explore structure/function studies. These are descriptive studies that are attempting to gain clues as to how the proteins carry out transport and are regulated. However, they fall short of defining how the proteins work, which presumably will follow from crystallagraphic techniques, although no mammalian transport protein has yet yielded the required information using any approach or combination of approaches.