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IRBIT functionally enhances the electroneutral Na + ‐coupled bicarbonate transporter NCBE by sequestering an N‐terminal autoinhibitory domain
Author(s) -
Parker Mark Daniel,
Daly Christopher,
Skelton LaraAnne,
Boron Walter
Publication year - 2007
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.21.6.a1285-b
Subject(s) - immunoprecipitation , chemistry , c terminus , exon , mutant , xenopus , green fluorescent protein , n terminus , microbiology and biotechnology , hek 293 cells , biophysics , biology , biochemistry , amino acid , peptide sequence , gene
IRBIT binds to and functionally enhances the electrogenic Na‐HCO 3 cotransporter NBCe1‐B (Shirakabe et al ., PNAS, 103, 2006). In an accompanying abstract, we further demonstrate that IRBIT functionally enhances the electroneutral Na‐coupled HCO 3 transporters NBCn1, NDCBE and NCBE. Taking the NCBE/IRBIT pair as our model, we co‐expressed NCBE with a C‐terminal EGFP tag (NCBE‐EGFP) and IRBIT with an N‐terminal HA‐tag (HA‐IRBIT) in Xenopus oocytes. The interaction was confirmed in oocytes by co‐immunoprecipitation of HA‐IRBIT (but not the S71A mutant known not to bind to NBCe1‐B) with an anti‐GFP antibody. We assayed the function of NCBE‐EGFP by exposing oocytes to a CO 2 /HCO 3 solution and monitoring the rate of the subsequent pH i recovery from the CO 2 ‐induced acid‐load using pH‐sensitive microelectrodes. NCBE‐EGFP activity was enhanced by co‐expression of HA‐IRBIT but not the S71A mutant. Truncating the N terminus of NCBE by removing protein encoded by exons 1 (residues 1‐16) or exons 1 and 2 (residues 1‐43), affected neither NCBE‐EGFP activity nor its enhancement by co‐expressed HA‐IRBIT. Removing the protein encoded by exons 1, 2 and 3 (residues 1‐92) doubled the rate of pH i recovery—compared to full‐length NCBE in the absence of co‐expressed HA‐IRBIT—and negated the enhancement in the presence of HA‐IRBIT. Our data suggest that exon 3 of NCBE encodes an autoinhibitory domain (AID), and that IRBIT enhances the activity of NCBE by sequestering the AID.