Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney

Abstract

We have used cDNA probes derived from the secretory form of the Na-K-Cl cotransporter to screen both cortical and medullary rabbit kidney cDNA libraries. A sequence of 4750 bases was identified from multiple clones. The DNA encodes a protein containing 1099 amino acids, which is 61% identical over its length to the secretory Na-K-Cl cotransporter from shark rectal gland. From analysis of amino acid hydropathy, we predict that this putative renal Na-K-Cl cotransporter has 12 transmembrane helices and large N- and C-terminal cytoplasmic regions. Two sites for N-linked glycosylation are predicted on an extracellular loop. Three potential sites for modulation by protein kinase A are in the C-terminal cytoplasmic domain. Most of the isolated renal cDNA clones were identical over all regions of overlap; however, there was a 96-bp region for which there were three different but homologous variants (A, B, and F). This region of divergence was identified as an alternatively spliced cassette exon since clones were identified that contained intronic DNA as well as consensus splice acceptor sites that bounded the region. Tissue Northern blot analysis revealed a broad band at approximately 5.1 kb that was unique to the kidney. High-stringency Northern blot analysis of cortical and medullary mRNA using antisense oligonucleotides synthesized over each of the three cassette exons revealed that the isoforms were differentially distributed within the kidney--B almost exclusively in cortex, F almost exclusively in medulla, and A about equally distributed.