Mechanism of CO ₂ and NH ₃ transport through human aquaporin 1: Evidence for parallel CO ₂ pathways

Abstract 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 CO 2 and NH 3 , whereas some aquaporin (AQP) water channels—tetramers with hydrophobic central pores—are permeable to CO 2 , NH 3 or both. Nevertheless, we understand neither the routes that CO 2 and NH 3 take through AQP tetramers, nor the basis of CO 2 /NH 3 selectivity. Here, we show—for human AQP1 (hAQP1)—that virtually all NH 3 and H 2 O pass through the hydrophilic, monomeric pores. However, CO 2 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 CO 2 or NH 3 influxes. We found that p‐chloromercuribenzene sulfonate (pCMBS)—which reacts with C189 in the monomeric pore—eliminates the entire hAQP1‐dependent (*) NH 3 signal (ΔpH S *) NH3 , but only half of the signals for CO 2 (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 CO 2 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 CO 2 /NH 3 moves through monomeric pores versus an alternate pathway (e.g., the central pore). image Key points Some membranes have negligible CO 2 permeability in the absence of protein channels like aquaporin‐1 (AQP1). We confirm that, during CO 2 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 NH 3 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 CO 2 traverses AQP1 via the monomeric pore plus a novel DIDS‐sensitive route that may be the central pore.