Exploring the CO2 permeability of cysteine‐less human aquaporin‐5 (hAQP5) with single introduced Cys residues

In the substituted cysteine accessibility method (SCAM), one introduces individual Cys residues into protein regions (e.g., binding sites or channel lumen) and then applies thiol (‐SH)‐specific reagents to the engineered proteins to determine whether they are functionally modified. Here we used microelectrodes to monitor the maximum rise in extracellular surface pH (ΔpH S ) caused by CO 2 influx into Xenopus oocytes (an index of CO 2 permeability), and monitored cell swelling under hypotonic conditions to compute osmotic water permeability (P f ). First, we mutated all 3 cysteines in hAQP5 to serine. The CO 2 and H 2 O permeabilities of this Cys‐less construct are similar to those of wt hAQP5. We then introduced single Cys residues back into the previously Cys‐less construct, targeting the extracellular loop that extends from the aquapore to the central pore (amino‐acid residues 32 to 44). We found that all such mutants—except L39C and I42C—lost their CO 2 permeability. Interestingly, most mutations maintained normal water permeability; only A32C, L33C, W35C and P36C had decreased P f vs wt. One possible explanation for the loss of CO 2 permeability in 11 of 13 mutants is that the newly introduced Cys residues form disulfide bonds between monomers. If so, we predict that treatment with a reducing agent (e.g., DTT) may break the disulfide bond and restore CO 2 permeability. Funded by AHA 09POST2060873 and ONR N00014 ‐08‐10532.