Evidence that DIDS crosslinks Aquaporin 1 monomers

Abstract

Our laboratory has shown that DIDS causes a ~60% decrease in the CO2 permeability (PCO2) 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 (AQP1FLAG) contributed the same H2O permeability (Pf), CO2-induced surface-pH (ΔpHS) spike, and DIDS sensitivity of this spike. Following SDS-PAGE, western-blot analysis (WBA) of AQP1FLAG (+/− 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 AQP1FLAG 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.