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Evidence that DIDS crosslinks Aquaporin 1 monomers
Author(s) -
Geyer R.Ryan,
MusaAziz Raif,
Boron Walter F.
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.1039.26
Subject(s) - dids , chemistry , western blot , aquaporin 1 , monomer , mutant , biochemistry , xenopus , microbiology and biotechnology , biophysics , membrane , biology , gene , water channel , organic chemistry , polymer , mechanical engineering , engineering , inlet
Our laboratory has shown that DIDS causes a ~60% decrease in the CO 2 permeability (P CO2 ) of wild‐type (wt) AQP1 and its C189S mutant. pCMBS inhibits AQP1 by ~40% but has no affect on C189S. DIDS + pCMBS inhibit AQP1 by ~100%. Thus, the two compounds act at different AQP1 locations. We hypothesize that DIDS (with 2 –NCS groups at opposite ends of the molecule) crosslinks two AQP1 monomers, thereby blocking the central pore. As expressed in Xenopus oocytes and compared with wt AQP1, FLAG‐tagged human AQP1 (AQP1 FLAG ) contributed the same H 2 O permeability (P f ), CO 2 ‐induced surface‐pH (ΔpH S ) spike, and DIDS sensitivity of this spike. Following SDS‐PAGE, western‐blot analysis (WBA) of AQP1 FLAG (+/− DIDS) purified from oocyte membranes showed that DIDS induces dimerization. LC/MS/MS of digested samples resulted in ~70% coverage of AQP1. To increase protein yield, we expressed AQP1 FLAG in P. pastoris , spheroplasted, and reacted with DIDS. After SDS‐PAGE of crude extract, WBA reveals that DIDS markedly increases AQP1 dimerization, consistent with the oocyte results. The remaining extract was affinity purified, separated by SDS‐PAGE, and stained with Deep Purple. Again, DIDS increased dimerization. Future studies will be aimed at identifying specific amino acids involved in DIDS crosslinking between AQP1 monomers.