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 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 both through 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 move through monomeric pores vs. an alternate pathway (e.g., the central pore).

Key points: Some membranes have little or no CO ₂ permeability, absent 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; (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 reduces (ΔpH _S *) _CO2 to zero. The C189S mutation of AQP1 eliminates the effects of pCMBS, but not 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.