Characterization and expression of plasma membrane Ca²⁺ ATPase (PMCA3) in the crayfish Procambarus clarkii antennal gland during molting

The discontinuous pattern of crustacean cuticular mineralization (the molting cycle) has emerged as a model system to study the spatial and temporal regulation of genes that code for Ca²⁺-transporting proteins including pumps, channels and exchangers. The plasma membrane Ca ²⁺-ATPase (PMCA) is potentially of significant interest due to its role in the active transport of Ca²⁺ across the basolateral membrane, which is required for routine maintenance of intracellular Ca²⁺ as well as unidirectional Ca²⁺ influx. Prior research has suggested that PMCA expression is upregulated during periods of elevated Ca²⁺ influx associated with postmolt cuticular mineralization. This paper describes the cloning, sequencing and functional characterization of a novel PMCA3 gene from the antennal gland (kidney) of the crayfish Procambarus clarkii. The complete sequence, the first obtained from a non-genetic invertebrate species, was obtained through reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. Crayfish PMCA3 consists of 4148 bp with a 3546 bp open reading frame coding for 1182 amino acid residues with a molecular mass of 130 kDa. It exhibits 77.5-80.9% identity at the mRNA level and 85.3-86.9% identity at the protein level with PMCA3 from human, mouse and rat. Membrane topography was typical of published mammalian PMCAs. Northern blot analysis of total RNA from crayfish gill, antennal gland, cardiac muscle and axial abdominal muscle revealed that a 7.5 kb species was ubiquitous. The level of PMCA3 mRNA expression in all tissues (transporting epithelia and muscle) increased significantly in pre/postmolt stages compared with relatively low abundance in intermolt. Western analysis confirmed corresponding changes in PMCA protein expression (130 kDa).