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Isoforms of the Na-K-2Cl cotransporter in murine TAL I. Molecular characterization and intrarenal localization
D B Mount1, A Baekgaard, A E Hall
1Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. david.mount@mcmail.vanderbilt.edu
Abstract:
We have identified several alternatively spliced cDNAs encoding mBSC1, an apical bumetanide-sensitive Na+-K+-2Cl- cotransporter from mouse kidney. Two full-length clones were isolated, designated C4 and C9, predicting proteins of 770 and 1,095 amino acids, respectively. The C4 isoforms are generated by utilization of an alternative polyadenylation site located within the intron between exons 16 and 17 of the mBSC1 gene on chromosome 2; the resultant transcripts predict a truncated COOH terminus ending in a unique 55 amino acid sequence. The predicted C4 and C9 COOH termini differ in the distribution of putative phosphorylation sites for both protein kinase A and C. Independent splicing events involve three previously described cassette exons, which are predicted to encode most of the second transmembrane domain. A total of six different isoforms are expressed, generated by the combinatorial association of three cassette exons and two alternative 3' ends. C9-specific and C4-specific antibodies detect proteins of approximately 150 and 120 kDa, respectively, in mouse kidney. Immunofluorescence and immunohistochemistry indicate expression of both COOH-terminal isoforms within the thick ascending limb of the loop of Henle (TAL). However, staining with the C4 antibody is more heterogeneous, with a decreased proportion of positive cells in the cortical TAL. Functional expression in Xenopus oocytes indicates a dominant negative function for C4 isoforms [companion study, C. Plata, D. B. Mount, V. Rubio, S. C. Hebert, and G. Gamba. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999], and the differential expression of these isoforms may contribute to functional heterogeneity of Na+-K+-2Cl- cotransport in mouse TAL.
Insights
Researchers identified mouse kidney Na+-K+-2Cl- cotransporter (mBSC1) isoforms C4 and C9. Differential splicing and alternative 3' ends generate six mBSC1 variants, impacting kidney function.
Area of Science:
- Molecular Biology
- Renal Physiology
- Biochemistry
Background:
- The Na+-K+-2Cl- cotransporter (BSC1) is crucial for renal salt reabsorption.
- Alternative splicing and alternative polyadenylation are key mechanisms for generating protein diversity.
Purpose of the Study:
- To identify and characterize alternatively spliced variants of the mouse apical Na+-K+-2Cl- cotransporter (mBSC1).
- To investigate the structural and functional differences between mBSC1 isoforms.
Main Methods:
- Isolation and sequencing of full-length cDNAs (C4 and C9).
- Analysis of alternative splicing and polyadenylation events.
- Protein expression analysis using isoform-specific antibodies and Western blotting.
- Immunofluorescence and immunohistochemistry for tissue localization.
- Functional expression studies in Xenopus oocytes.
Main Results:
- Two full-length mBSC1 clones (C4 and C9) were identified, predicting proteins of 770 and 1,095 amino acids.
- Six mBSC1 isoforms result from combinatorial splicing of three cassette exons and two alternative 3' ends.
- Isoforms C4 and C9 exhibit distinct COOH termini with different phosphorylation site distributions.
- Western blotting detected C4 (~120 kDa) and C9 (~150 kDa) proteins in mouse kidney.
- Immunohistochemistry localized both isoforms to the thick ascending limb of the loop of Henle (TAL), with heterogeneous C4 expression.
- Functional studies showed C4 isoforms possess dominant-negative activity.
Conclusions:
- Differential expression of mBSC1 isoforms, generated by alternative splicing and polyadenylation, contributes to functional heterogeneity in mouse TAL.
- The distinct structural features and functional properties of mBSC1 isoforms may play a role in regulating renal salt transport.