Basolateral K-Cl cotransporter regulates colonic potassium absorption in potassium depletion.

Active potassium absorption in the rat distal colon is electroneutral, Na(+)-independent, partially chloride-dependent, and energized by an apical membrane H,K-ATPase. Both dietary sodium and dietary potassium depletion substantially increase active potassium absorption. We have recently reported that sodium depletion up-regulates H,K-ATPase alpha-subunit mRNA and protein expression, whereas potassium depletion up-regulates H,K-ATPase beta-subunit mRNA and protein expression. Because overall potassium absorption is non-conductive, K-Cl cotransport (KCC) at the basolateral membrane may also be involved in potassium absorption. Although KCC1 has not been cloned from the colon, we established, in Northern blot analysis with mRNA from the rat distal colon using rabbit kidney KCC1 cDNA as a probe, the presence of an expected size mRNA in the rat colon. This KCC1 mRNA is substantially increased by potassium depletion but only minimally by sodium depletion. KCC1-specific antibody identified a 155-kDa protein in rat colonic basolateral membrane. Potassium depletion but not sodium depletion resulted in an increase in KCC1 protein expression in basolateral membrane. The increase of colonic KCC1 mRNA abundance and KCC1 protein expression in potassium depletion of the rat colonic basolateral membrane suggests that K-Cl cotransporter: 1) is involved in transepithelial potassium absorption and 2) regulates the increase in potassium absorption induced by dietary potassium depletion. We conclude that active potassium absorption in the rat distal colon involves the coordinated regulation of both apical membrane H,K-ATPase and basolateral membrane KCC1 protein.

The mouse and human genes encoding the recognition component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.