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SPAK isoforms differentially regulate renal sodium transport
Author(s) -
McCormick James Arthur,
Delpire Eric,
Ellison David H
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1038.23
Subject(s) - distal convoluted tubule , gene isoform , kinase , exon , cotransporter , chemistry , microbiology and biotechnology , knockout mouse , western blot , phosphorylation , symporter , downregulation and upregulation , kidney , biology , biochemistry , transporter , endocrinology , gene , sodium , nephron , organic chemistry
SPAK is a protein kinase that phosphorylates and activates the cation chloride cotransporters NCC, NKCC2 and NKCC1. SPAK knockout (KO) mice display a phenotype consistent with NCC deletion. Total and phospho‐NCC protein abundance is lower in SPAK KO mice; surprisingly, phospho‐NKCC2, but not total NKCC2, is increased. We examined the mechanism causing the discordant effects of SPAK on NKCC2 and NCC. Western blot analysis with a C‐terminal antibody showed a single SPAK isoform in brain (70KDa), but multiple SPAK isoforms in kidney (70KDa, 60KDa and 46KDa). An N‐terminal antibody detected 70KDa SPAK in brain and kidney, but 60KDa and 46KDa SPAK isoforms were not detected. Immunofluorescence showed that only full‐length SPAK co‐localized with NCC at the convoluted tubule (DCT); in the thick ascending limb (TAL), the site of NKCC2 expression, the majority of SPAK lacks the N‐terminus. RACE PCR of kidney RNA identified a novel cDNA with an alternative first exon, followed by exon 6. The 46KDa (exon 6 START) and 60KDa (alternative exon 1 START) SPAK forms lack the PAPA box and parts of the kinase domain. We propose that normally, kinase‐deficient SPAK suppresses NKCC2 activity in the TAL through a dominant‐negative on full‐length SPAK and the related kinase OSR1. Deletion of SPAK relieves OSR1 inhibition, resulting in NKCC2 activation. In the DCT, where only full‐length SPAK is expressed, deletion causes inhibition of NCC.