Cloning, embryonic expression, and alternative splicing of a murine
kidney-specific na-k-cl cotransporter (nkcc2).
Igarashi, Peter, Gregory B. Vanden Heuvel, John A. Payne, and Bliss
Forbush, Iii.
Departments of Internal Medicine and Cellular and Molecular
Physiology, Yale University School of Medicine, 333 Cedar Street, New
Haven, CT 06520
APStracts 2:0055F, 1995.
A full-length cDNA encoding the murine renal Na-K-Cl cotransporter
(NKCC2) was cloned using library screening and anchored PCR. The
deduced protein sequence contained 1,095 amino acids and was 93.5%
identical to rabbit NKCC2 and 97.6% identical to rat BSC1. Two
potential sites of phosphorylation by cAMP-dependent protein kinase
and 7 potential sites of phosphorylation by protein kinase C which
were previously identified in the rabbit and rat sequences were
phylogenetically conserved in the mouse. The expression of NKCC2 in
the mouse was examined using northern blot analysis and in situ
hybridization. Expression of NKCC2 was kidney-specific in both adult
and embryonic mice. In the developing metanephros, NKCC2 was induced
at 14.5 days p.c. and was expressed in distal limbs of immature loops
of Henle but was absent from the ureteric bud, S-shaped bodies, and
earlier nephrogenic structures. Similar to the rabbit, isoforms of
NKCC2 that differed in the sequence of a 96-bp segment were
identified in the mouse. In situ hybridization revealed that the
isoforms exhibited different patterns of expression in the mature
thick ascending limb of the loop of Henle: Isoform F was most highly
expressed in the inner stripe of outer medulla, isoform A was most
highly expressed in the outer stripe of the outer medulla, and
isoform B was most highly expressed in the cortical thick ascending
limb. To verify that the isoforms were generated by alternative
splicing of mutually exclusive cassette exons, genomic clones
encoding murine NKCC2 were characterized. Cassette exons were
identified that corresponded to each of the three isoforms and were
flanked by consensus splice donor and acceptor sequences.
Received 23 January 1995; accepted in final form 10 April 1995.
APS Manuscript Number F20-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 25 April 1995.