Cloning and molecular characterisation of the trout (Oncorhynchus mykiss) vacuolar H(+)-ATPase B subunit.

The current model of transepithelial ion movements in the gill of freshwater fish incorporates an apically oriented vacuolar H(+)-ATPase (H(+)V-ATPase; proton pump) that is believed to facilitate both acid excretion and Na(+) uptake. To substantiate this model, we have cloned and sequenced a cDNA encoding the B subunit of the rainbow trout (Oncorhynchus mykiss) H(+)V-ATPase. The cloning of the B subunit enabled an examination by northern analysis of its tissue distribution and expression during external hypercapnia. Degenerate oligonucleotide primers to the B subunit of the H(+)V-ATPase were designed and used in a semi-nested polymerase chain reaction (PCR) to amplify an 810 base pair (bp) product from a trout gill/kidney cDNA library. This PCR product was cloned and sequenced and then used to screen the same cDNA library. The assembled 2262 bp cDNA included an open reading frame coding for a deduced protein of 502 amino acid residues. A BLAST search of the GenBank nucleotide database revealed numerous matches to other vertebrate and invertebrate H(+)V-ATPase B subunits. Protein alignment demonstrated that the trout H(+)V-ATPase B subunit is more than 85 % identical and more than 90 % similar to those in other vertebrate species. An initial analysis of H(+)V-ATPase mRNA tissue distribution revealed significant expression in blood. Although a comparison of perfused tissues (blood removed) with non-perfused tissues demonstrated no obvious contribution of the blood to total tissue H(+)-ATPase mRNA levels, all subsequent experiments were performed using perfused tissues. Levels of H(+)V-ATPase mRNA expression were high in the gill, kidney (anterior or posterior), intestine, heart and spleen, but lower in liver and white muscle. Exposure of the fish to 12 h of external hypercapnia (water P(CO2)=7. 5 mmHg; 1 kPa) was associated with a transient increase (at 2 h) in the levels of H(+)V-ATPase B subunit mRNA in gill and kidney; liver mRNA levels were unaffected. These results are consistent with the hypothesis of an apically localised plasma membrane H(+)V-ATPase in the freshwater trout gill and that the expression of this proton pump is increased during periods of acidosis, at least in part because of an increased steady-state level of H(+)V-ATPase mRNA.

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