Synergistic roles of Aquaporin 5 and Intra- and Extracellular Carbonic Anhydrases in promoting CO ₂ Diffusion across the Xenopus Oocyte Plasma Membrane

Key Points

• According to Fick’s law, transmembrane CO ₂ flux ( J CO ₂ ) is the product of membrane permeability ( P _M,CO2 ) and transmembrane concentration gradient (Δ[CO ₂ ]): J CO ₂ = P _M,CO2 Δ[CO ₂ ]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhances P _M,CO2 , and (2) intracellular and (3) extracellular carbonic anhydrases (CAs) enhance Δ[CO ₂ ] by consuming accumulated or replenishing lost CO ₂ . We now examine interactio ns among #1–#3.

• We assess CO ₂ fluxes—produced by addition/removal of extracellular CO ₂ / —using microelectrodes to monitor extracellular-surface pH (pH _S ) and intracellular pH (pH _i ) of Xenopus oocytes heterologously expressing hAQP5, injected with human CA II (hCA II), and/or exposed to extracellular bovine CA (bCA).

• Enhancing effects on CO ₂ fluxes are synergistic among hAQP5, hCA II, and bCA, any of which can become rate limiting, depending on the status of the other two.

• CO ₂ / addition transiently increases pH _S (ΔpH _S ), hCA II augments ΔpH _S (ΔΔpH _S ), and hAQP5 enhances ΔΔpH _S (ΔΔΔpH _S )—a novel tool to assess potential CO ₂ channels.

CO ₂ diffusion across plasma membranes depends on both membrane CO ₂ permeability ( P _M,CO2 ) and transmembrane CO ₂ concentration gradient (Δ[CO ₂ ])—Fick’s law. Human aquaporin-5 (hAQP5) accelerates CO ₂ diffusion by increasing P _M,CO2 , whereas carbonic anhydrases (CAs) accelerate CO ₂ diffusion by enhancing CO ₂ consumption/production and thus Δ[CO ₂ ]. Here, we systematically assess functional interactions among a gas channel and intra-/extracellular CAs. On Day 1, we inject Xenopus oocytes with cRNA encoding hAQP5 (control: H ₂ O). On Day 4, we inject hCA II protein in “Tris” buffer (control: “Tris”). We assess CO ₂ fluxes by introducing extracellular 1.5% CO ₂ /10 mM and using microelectrodes to measure (1) maximal extracellular-surface pH increase ΔpH _S , (2) maximal rate of pH _S relaxation (dpH _S /dt) _Max , and (3) maximal rate of intracellular-pH decrease (dpH _i /dt) _Max . By itself, hCA II minimally increases ΔpH _S —measured “trans” to added cytosolic CA (CA _i )—even at highest doses (100 ng/oocyte). However, hAQP5 alone triples ΔpH _S , an effect further doubled by increasing hCA II. By itself, bovine erythrocyte CA (bCA) in the extracellular fluid doubles (dpH _i /dt) _Max magnitude—meas ured “trans” to added extracellular CA (CA _o )—an effect further doubled by hAQP5. Note: pH measureme nts “cis” to added CAs—pH _S for bCA, (dpH _i /dt) _Max for hCA II—are overwhelmed by enzymatical ly-produced/consumed H ⁺ , and cannot provide intuitive insight into CO ₂ fluxes. Our “trans” pH measurements: (1) confirm synergy between CA _o and CA _i ; establish synergy between hAQP5 and both (2) CA _o and (3) CA _i ; and show that enhancement of ΔpH _S by CA _i (ΔΔpH _S ) is a useful tool for assessing CO ₂ permeability of membrane proteins (e.g., hAQP5).