Molecular cloning and tissue distribution of crayfish plasma membrane calcium ATPase

Calcium is critical to cell function. The basolateral Plasma Membrane Ca2+ ATPase (PMCA) plays an important role in regulating the intracellular Ca2+ concentration. This is especially true during the postmolt stage of the molting cycle when whole animal influx of Ca occurs. The purpose of this work was to clone and characterize PMCA from crayfish Procambarus clarkii. Based on the conserved DNA sequence of rat PMCA, two degenerate primers 5′GCIT-CIGAYATHATHCTIAC3′ and 5′GCRTTDATYTCRTTRAAIAG3′ were used to PCR double stranded cDNA prepared from crayfish heart and muscle. A 497 bp PCR product (corresponding to nucleotide sequence 2598 - 3455 of PMCA isoform 2 in rat) was cloned and sequenced. A genbank search for nucleotide homology revealed that the crayfish sequence shares 64-69% identity with PMCA from rat, human, pig and rabbit. The deduced amino acid sequence shares 67-70% identity with those PMCAs. Northern blot of intermolt crayfish mRNA from heart, liver, gills, unfertilized eggs, muscle and kidney was probed with this 497 bp partial PMCA sequence. The probe hybridized with a 7 kb fragment in all six tissues. The signals in eggs and kidney were much stronger than that in the other four tissues indicating a more active role of the gene. There is an additional 5 kb band in the eggs indicating a possible isoform or precursor.