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 ${\text{CO}}_2$ and ${\text{NH}}_3$ , whereas some aquaporin (AQP) water channels-tetramers with hydrophobic central pores-are permeable to ${\text{CO}}_2,{\text{NH}}_3$ , or both. Nevertheless, we understand neither the routes that ${\text{CO}}_2$ and ${\text{NH}}_3$ take through AQP tetramers, nor the basis of ${\text{CO}}_2/{\text{NH}}_3$ selectivity. Here, we show-for human AQP1 (hAQP1)-that virtually all ${\text{NH}}_3$ and ${\text{H}}_2\text{O}$ pass through the hydrophilic, monomeric pores. However ${\text{CO}}_2$ 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 $\left(\Delta {\text{pH}}_{\text{S}}\right)$ caused by ${\text{CO}}_2$ or ${\text{NH}}_3$ influxes. We found that p-chloromercuribenzene sulfonate (pCMBS)-which reacts with C189 in the monomeric pore-eliminates the entire hAQP1-dependent $(*){\text{NH}}_3$ signal ${\left({{\Delta \text{pH}}_{\text{S}}}^{*}\right)}_{{\text{NH}}_3}$ , but only half of the signals for ${\text{CO}}_2{\left({{\Delta \text{pH}}_{\text{H}}}^{*}\right)}_{{\text{CO}}_2}$ or osmotic water permeability $P_{\text{f}}\text{*}$ 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), eliminates the remaining ${\left({{\Delta \text{pH}}_{\text{S}}}^{*}\right)}_{{\text{CO}}_2}$ but has no effect on ${\left({{\Delta \text{pH}}_{\text{S}}}^{*}\right)}_{{\text{NH}}_3}$ or $P_{\text{f}}\text{*}$ Together, the two drugs completely eliminate the ${\text{CO}}_2$ 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 ${\text{CO}}_2/{\text{NH}}_3$ move through monomeric pores vs. an alternate pathway (e.g., the central pore).