Effect of DIDS and pCMBS on the CO2 permeability of human aquaporin‐5 (AQP5)

Abstract

Recent work from our laboratory showed that AQP1, AQP4, and AQP5 each exhibits a characteristic ratio for indices of CO2 vs. NH3 permeability. However, the mechanism of CO2 transport is not well understood. Here, we used microelectrodes to monitor the maximum rise in extracellular surface pH (ΔpHS) caused by CO2 influx into Xenopus oocytes, and monitored cell swelling under hypotonic conditions to compute osmotic water permeability (Pf). Subtracting values of H2O‐injected oocytes from those expressing FLAG‐tagged human (h) AQP5 yielded hAQP5‐dependent (*) ΔpHS or Pf. Treating FLAG‐hAQP5 oocytes with 100 μM DIDS × 60 min decreased ΔpHS* by 80±23%, similar to the observation of Musa‐Aziz et al on rat AQP5 (2007 JASN abstract). The DIDS inhibition for FLAG‐hAQP5 was not different from 61±12% for FLAG‐hAQP5‐C182S (eliminating potential DIDS and pCMBS target) or from 66±16% for FLAG‐hAQP5‐K34N (eliminating potential DIDS target). DIDS did not affect Pf* for any construct. Treating with 1 mM pCMBS × 30 min had no effect on ΔpHS* for any construct. pCMBS modestly decreased Pf* for WT and K34N, but not for C182S. In conclusion, DIDS markedly reduces the CO2 but not H2O permeability of AQP5, whereas pCMBS reduces the H2O but not CO2 permeability. Thus, our data are consistent with the hypothesis that the major route for CO2 through AQP5 is not the monomeric aquapore. One candidate is the central pore between the four monomers.