Synergistic roles of Aquaporin 5 and Intra- and Extracellular Carbonic Anhydrases in promoting CO₂Diffusion across the Xenopus Oocyte Plasma Membrane

Key Points

• According to Fick’s law, transmembrane CO₂flux (JCO₂) is the product of membrane permeability (P _M,CO2) and transmembrane concentration gradient (Δ[CO₂]):JCO₂=P _M,CO2Δ[CO₂]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhancesP _M,CO2, and (2) intracellular and (3) extracellular carbonic anhydrases (CAs) enhance Δ[CO₂] by consuming accumulated or replenishing lost CO₂. We now examine interactio ns among #1–#3.

• We assess CO₂fluxes—produced by addition/removal of extracellular CO₂/<inline-formula> <inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="647833v2_inline1.gif"/> </inline-formula>—using microelectrodes to monitor extracellular-surface pH (pH_S) and intracellular pH (pH_i) ofXenopusoocytes heterologously expressing hAQP5, injected with human CA II (hCA II), and/or exposed to extracellular bovine CA (bCA).

• Enhancing effects on CO₂fluxes are synergistic among hAQP5, hCA II, and bCA, any of which can become rate limiting, depending on the status of the other two.

• CO₂/<inline-formula> <inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="647833v2_inline2.gif"/> </inline-formula>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 CO₂channels.

CO₂diffusion across plasma membranes depends on both membrane CO₂permeability (P _M,CO2) and transmembrane CO₂concentration gradient (Δ[CO₂])—Fick’s law. Human aquaporin-5 (hAQP5) accelerates CO₂diffusion by increasingP _M,CO2, whereas carbonic anhydrases (CAs) accelerate CO₂diffusion by enhancing CO₂consumption/production and thus Δ[CO₂]. Here, we systematically assess functional interactions among a gas channel and intra-/extracellular CAs. On Day 1, we injectXenopusoocytes with cRNA encoding hAQP5 (control: H₂O). On Day 4, we inject hCA II protein in “Tris” buffer (control: “Tris”). We assess CO₂fluxes by introducing extracellular 1.5% CO₂/10 mM<inline-formula> <inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="647833v2_inline3.gif"/> </inline-formula>and using microelectrodes to measure (1) maximal extracellular-surface pH increase ΔpH_S, (2) maximal rate of pH_Srelaxation (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)_Maxmagnitude—meas ured “trans” to added extracellular CA (CA_o)—an effect further doubled by hAQP5. Note: pH measureme nts “cis” to added CAs—pH_Sfor bCA, (dpH_i/dt)_Maxfor hCA II—are overwhelmed by enzymatical ly-produced/consumed H⁺, and cannot provide intuitive insight into CO₂fluxes. Our “trans” pH measurements: (1) confirm synergy between CA_oand CA_i; establish synergy between hAQP5 and both (2) CA_oand (3) CA_i; and show that enhancement of ΔpH_Sby CA_i(ΔΔpH_S) is a useful tool for assessing CO₂permeability of membrane proteins (e.g., hAQP5).