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 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 both through the monomeric pores and another pathway. We expressed hAQP1 in Xenopus oocytes and used microelectrodes to monitor the maximal surface-pH transient ({Delta}pHS) 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 ({Delta}pHS*)NH3, but only half of the signals for CO2 ({Delta}pHS*)CO2 or osmotic water permeability Pf*. 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS), eliminates the remaining ({Delta}pHS*)CO2 but has no effect on ({Delta}pHS*)NH3 or Pf*. 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 move through monomeric pores vs. an alternate pathway (e.g., the central pore). Key PointsO_LISome membranes have little or no CO2 permeability, absent protein channels like aquaporin-1 (AQP1). C_LIO_LIWe 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 ({Delta}pHS*)CO2, measured with microelectrodes. During NH3 influx, hAQP1 expression increases the magnitude of the transient pHS decrease by ({Delta}pHS*)NH3. C_LIO_LIp-chloromercuribenzene sulfonate (pCMBS), which reacts with C189 in the monomeric pore, reduces ({Delta}pHS*)CO2 by; ({Delta}pHS*)NH3, to zero; and AQP1-dependent osmotic water permeability (Pf*), by half. C_LIO_LI4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS) reduces ({Delta}pHS*)CO2 by half, but has no effect on ({Delta}pHS*)NH3 or Pf*. DIDS crosslinks AQP1 monomers expressed in Pichia pastoris. C_LIO_LITogether, pCMBS+DIDS reduces ({Delta}pHS*)CO2 to zero. The C189S mutation of AQP1 eliminates the effects of pCMBS, but not 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. C_LI