Mechanism of CO₂ and NH₃ transport through human aquaporin 1: Evidence for parallel CO₂ pathways

The traditional view had been that dissolved gases cross membranes simply by dissolving in and diffusing through the membrane lipid. However, some membranes are impermeable to CO₂ and NH₃, whereas some aquaporin (AQP) water channels-tetramers with hydrophobic central pores-are permeable to CO₂, NH₃ or both. Nevertheless, we understand neither the routes that CO₂ and NH₃ take through AQP tetramers, nor the basis of CO₂/NH₃ selectivity. Here, we show-for human AQP1 (hAQP1)-that virtually all NH₃ and H₂O pass through the hydrophilic, monomeric pores. However, CO₂ passes through both the monomeric pores and another pathway. We expressed hAQP1 in Xenopus oocytes and used microelectrodes to monitor the maximal surface-pH transient (ΔpH_S) caused by CO₂ or NH₃ influxes. We found that p-chloromercuribenzene sulfonate (pCMBS)-which reacts with C189 in the monomeric pore-eliminates the entire hAQP1-dependent (*) NH₃ signal (ΔpH_S*)_NH3, but only half of the signals for CO₂ (ΔpH_S*)_CO2 or osmotic water permeability P_f*. 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), eliminates the remaining (ΔpH_S*)_CO2 but has no effect on (ΔpH_S*)_NH3 or P_f*. Together, the two drugs completely eliminate the CO₂ permeability of hAQP1. When we express hAQP1 in Pichia pastoris, treat spheroplasts with DIDS and examine hAQP1 by SDS-PAGE, reactivity with an anti-DIDS antibody shows that DIDS crosslinks hAQP1 monomers. Our results provide the first evidence that a molecule can move through an AQP via a route other than the monomeric pore, and raise the possibility that selectivity depends on the extent to which CO₂/NH₃ moves through monomeric pores versus an alternate pathway (e.g., the central pore). KEY POINTS: Some membranes have negligible CO₂ permeability in the absence of protein channels like aquaporin-1 (AQP1). We confirm that, during CO₂ influx, heterologous expression of human AQP1 (hAQP1) in Xenopus oocytes increases the magnitude of the transient surface-pH increase by an amount (ΔpH_S*)_CO2, measured with microelectrodes. During NH₃ influx, hAQP1 expression increases the magnitude of the transient pH_S decrease by (ΔpH_S*)_NH3. p-chloromercuribenzene sulfonate (pCMBS), which reacts with C189 in the monomeric pore, reduces (ΔpH_S*)_CO2 by half; (ΔpH_S*)_NH3, to zero; and AQP1-dependent osmotic water permeability (P_f*), by half. 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) reduces (ΔpH_S*)_CO2 by half, but has no effect on (ΔpH_S*)_NH3 or P_f*. DIDS crosslinks AQP1 monomers expressed in Pichia pastoris. Together, pCMBS+DIDS reduce (ΔpH_S*)_CO2 to zero. The C189S mutation of AQP1 eliminates the effects of pCMBS, but not of DIDS. Our results thus show that CO₂ traverses AQP1 via the monomeric pore plus a novel DIDS-sensitive route that may be the central pore.