Identification of two partners from the bacterial Kef exchanger family for the apical plasma membrane V-ATPase of Metazoa

Abstract

The vital task of vectorial solute transport is often energised by a plasma membrane, proton-motive V-ATPase. However, its proposed partner, an apical alkali-metal/proton exchanger, has remained elusive. Here, both FlyAtlas microarray data and in situ analyses demonstrate that the bacterial kefB and kefC (members of the CPA2 family) homologues in Drosophila, CG10806 and CG31052, respectively, are both co-expressed with V-ATPase genes in transporting epithelia. Immunocytochemistry localises endogenous CG10806 and CG31052 to the apical plasma membrane of the Malpighian (renal) tubule. YFP-tagged CG10806 and CG31052 both localise to the plasma membrane of Drosophila S2 cells, and when driven in principal cells of the Malpighian tubule, they localise specifically to the apical plasma membrane. V-ATPase-energised fluid secretion is affected by overexpression of CG10806, but not CG31052; in the former case, overexpression causes higher basal rates, but lower stimulated rates, of fluid secretion compared with parental controls. Overexpression also impacts levels of secreted Na+ and K+. Both genes rescue exchanger-deficient (nha1 nhx1) yeast, but act differently; CG10806 is driven predominantly to the plasma membrane and confers protection against excess K+, whereas CG31052 is expressed predominantly on the vacuolar membrane and protects against excess Na+. Thus, both CG10806 and CG31052 are functionally members of the CPA2 gene family, colocalise to the same apical membrane as the plasma membrane V-ATPase and show distinct ion specificities, as expected for the Wieczorek exchanger.

Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities

“…The online atlas of gene expression, FlyAtlas.org, shows that both Nha1 and Nha2 are widely expressed, but particularly in the epithelia of the alimentary canal (15,23,24). To validate this pattern of gene expression, we performed quantitative real-time PCR, which demonstrated consistent enrichment levels at least as high as those seen in the Affymetrix-derived FlyAtlas (Fig.…”

“…Consistent with this, overexpression of GFP-tagged Nha1 or Nha2 labels the same apical membrane as the V-ATPase in the Malpighian (renal) tubule, a model epithelium in which transport and control mechanisms are conveniently studied (21,22), and RNAi against one of the exchangers affects fluid secretion (15). Thus, Drosophila provides an ideal system in which to investigate the roles of NHAs in multicellular animals.…”

“…The NHA family is relatively divergent, however, and asserting orthology between human and insect NHAs is difficult (5); in terms of sequence identity, insect NHA1 is slightly more similar to human NHA2, and vice versa. Both Drosophila NHAs are widely expressed but at particularly high levels in epithelia, suggesting a role in organismal ion homeostasis (15,16). Given that insect epithelia are energized by an apical plasma membrane H + V-ATPase (17,18), the NHAs have been hypothesized to act as the partner exchangers, as predicted by Wieczorek and coworkers (17,19), that colocalize with V-ATPase and use the proton electrochemical gradient to achieve transepithelial transport of sodium and potassium, similar to the position in mammalian MDCK cells (20).…”

Insect capa neuropeptides impact desiccation and cold tolerance

“…Capa increases V-ATPase activity by elevating mitochondrial calcium and ATP levels (38), increasing tubule diuresis. V-ATPase sets up a proton gradient, which then drives Na + and K + excretion through one or more exchangers such as sodium/proton exchanger (NHA) 1 and 2 (39). Thus, we propose that activation of capa signaling mediates recovery from nonlethal chill coma either by minimizing ion and water dysregulation during cold or by stimulating the redistribution of ions and water via the diuretic action of capa peptides on the tubule during recovery, or by both mechanisms (12).…”

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…In conjunction with the V-ATPase, it has been proposed that they secrete cations into the tubular lumen. This close coupling of a primary VATPase with a secondarily active cation/H + exchange has been refered to as the "Wieczorek model" [36,175].…”