Osmoregulation by gills of euryhaline crabs: Molecular analysis of transporters

American Zoologist, Sep 2001 by Towle, David W, Weihrauch, Dirk

A second ATP-utilizing pump, the vacuolar-type H^sup ^-ATPase, is considered to play an important role in ion uptake by a variety of animal species that successfully tolerate freshwater (Wieczorek et al., 1999). The Vtype ATPase is a highly conserved multisubunit protein similar to the F-type ATP synthase of mitochondria and chloroplasts. Found in eukaryotes, eubacteria, and archaea, the V-type ATPase transports H ions against an electrochemical gradient across vacuolar, vesicular, or plasma membranes (reviewed by Nelson et al., 2000). A plasma membrane form of the V-type ATPase has been implicated in energizing NaCl uptake by frog skin, freshwater teleost gill, and gill of the freshwater-tolerant Chinese crab Eriocheir sinensis (Lin and Randall, 1995; Onken and Putzenlechner, 1995; Ehrenfeld and Klein, 1997), where generation of a membrane potential may link its action to current flow through apical Na^sup ^ channels or basolateral Cl^sup -^ channels. The significance of the V-type ATPase in brackish-water-tolerant species is less clear.

Although these ATPases likely provide the electrochemical gradients that drive osmoregulatory ion transport, they must be accompanied by other transporters to complete the process. The Na^sup ^ K^sup ^-ATPase is exclusively located in the basolateral membrane of gill epithelial cells (Towle and Kays, 1986), where it likely pumps three Na^sup ^ ions outward toward the hemolymph and two K^sup ^ (or NH^sub 4^^sup ^) ions inward for each ATP hydrolyzed (Towle and Holleland, 1987). Thus the net movement of Na^sup ^ mediated by Na^sup ^ K^sup -^-ATPase is from cytosol to hemolymph, resulting in osmoregulatory accumulation of Na^sup ^ (and Cl-, by other mechanisms). Other transporters must mediate movement of Na^sup ^ ions from the environment across the apical membrane to the cytosol.

Transporters that may be involved in the apical uptake of Na^sup ^ and other ions from the environment include several likely candidates (Fig. 4). One of these is the Na^sup ^/H^sup ^ exchanger, a transmembrane protein found in many animal cells, where it moves Na^sup ^ across the plasma membrane into the cytosol in exchange for H^sup ^, depending on a chemical gradient of one of the ions (reviewed by Wakabayashi et al., 1997). Six isoforms of the Na^sup ^/H^sup ^ exchanger have been described in mammalian species, varying in their tissue and cellular locations and responses to regulatory factors. In gills of teleosts, Na^sup ^/H^sup ^ exchangers appear to be similar to those of other vertebrates, namely exchanging one Na for one H^sup ^ (Claiborne et al., 1999). In crustacean gills, however, a Na^sup ^/H^sup ^ exchanger has been identified that is electrogenic, apparently exchanging two Na^sup ^ for one H^sup ^(Shetlar and Towle, 1989). A similar electrogenic exchanger has been described in lobster and prawn hepatopancreas (Ahearn and Clay, 1989; Ahearn et al., 1990). Amiloride, an inhibitor of the Na^sup ^/H^sup -^ exchanger and epithelial Na channels, is known to reduce Na^sup ^ uptake across isolated crab gills (Burnett and Towle, 1990) (Fig. 3). Thus the Na^sup ^/H^sup ^ exchanger (or epithelial Na channel) represents one candidate possibly participating in the transfer of Na ions across the apical membrane. Because no evidence exists for epithelial Na channels in crustacean gills, we have focused our attention on the exchanger.