Gill-speciric transcriptional regulation of Na+/K+-ATPase alpha-subunit in the euryhaline shore crab Pachygrapsus marmoratus: sequence variants and promoter structure

Abstract

The sodium pump (Na+/K+-ATPase) has been implicated in osmoregulatory ion transport in many aquatic animals. In the euryhaline hyper-hypoosmoregulating shore crab Pachygrapsus marmoratus, induction of Na+/K+-ATPase alpha-subunit mRNA varies between gills in response to osmotic stress. Following transfer of crabs from normal seawater (36 parts per thousand salinity) to diluted seawater (10 parts per thousand), a condition in which gills exhibit net ion uptake, a-subunit mRNA expression is upregulated in all tested gills, albeit with differing time courses. By contrast, following transfer from seawater to hypertonic (45 parts per thousand) seawater, a condition in which the animal is excreting ions, alpha-subunit mRNA is induced primarily in gill no. 7 (nine in total), suggesting that this gill may be associated specifically with ion excretion in P. marmoratus. Full-length sequencing of alpha-subunit cDNA revealed the existence of two isoforms differing only in the inclusion of an 81-nucleotide segment within the N-terminal open reading frame of the long (D) form in comparison to the short (C) form. The 81-nucleotide segment encodes a 14-33 protein binding site that may facilitate movement of the alpha-subunit protein between intracellular compartments and the plasma membrane. mRNA expression of the two forms followed similar patterns upon salinity transfer. Genomic DNA sequencing of the putative promoter region of the alpha-subunit gene demonstrated a spectrum of predicted transcription factor binding sites that are likely associated with the complex expression pattern observed among gills following osmotic stress.