Synergistic roles of aquaporin 5 and intra‐ and extracellular carbonic anhydrases in promoting CO ₂ diffusion across the <i>Xenopus</i> oocyte plasma membrane

CO₂ diffusion across plasma membranes depends on both membrane CO₂ permeability ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>P</mml:mi> <mml:mrow><mml:mrow><mml:mi>M</mml:mi> <mml:mo>,</mml:mo> <mml:mi>C</mml:mi></mml:mrow> <mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> </mml:mrow> </mml:msub> <mml:annotation>${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$</mml:annotation></mml:semantics> </mml:math> ) and the transmembrane CO₂ concentration gradient (Δ[CO₂]) - Fick's law. Human aquaporin-5 (hAQP5) enhances CO₂ diffusion by increasing <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>P</mml:mi> <mml:mrow><mml:mrow><mml:mi>M</mml:mi> <mml:mo>,</mml:mo> <mml:mi>C</mml:mi></mml:mrow> <mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> </mml:mrow> </mml:msub> <mml:annotation>${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$</mml:annotation></mml:semantics> </mml:math> , whereas carbonic anhydrases (CAs) do so by enhancing CO₂ consumption/production and thus Δ[CO₂]. 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: H₂O). On Day 4 we inject hCA II protein in 'Tris' buffer (control: 'Tris'). We assess CO₂ fluxes by introducing extracellular 1.5% CO₂/10 mM HCO₃ ^- and using microelectrodes to measure (1) the maximal increase of extracellular surface pH (ΔpH_S), (2) the maximal rate of pH_S relaxation (dpH_S/dt)_Max and (3) the maximal rate of intracellular pH decrease (dpH_i/dt)_Max. By itself hCA II minimally increases ΔpH_S - measured on the side of the membrane opposite to the added cytosolic CA (CA_i) - even at our 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 - measured on the side of the membrane opposite to the added extracellular CA (CA_o) - an effect further doubled by hAQP5. Our 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 (4) show that the ability of CA_i to enhance ΔpH_S is a useful tool for assessing the CO₂ permeability of membrane proteins (e.g. hAQP5). KEY POINTS: According to Fick's law transmembrane CO₂ flux ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>J</mml:mi> <mml:mrow><mml:mi>C</mml:mi> <mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> </mml:mrow> </mml:msub> <mml:annotation>${J_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$</mml:annotation></mml:semantics> </mml:math> ) is the product of membrane permeability ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>P</mml:mi> <mml:mrow><mml:mrow><mml:mi>M</mml:mi> <mml:mo>,</mml:mo> <mml:mi>C</mml:mi></mml:mrow> <mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> </mml:mrow> </mml:msub> <mml:annotation>${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$</mml:annotation></mml:semantics> </mml:math> ) and transmembrane concentration gradient (Δ[CO₂]): <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>J</mml:mi> <mml:mrow><mml:mi>C</mml:mi> <mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> </mml:mrow> </mml:msub> <mml:annotation>${J_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$</mml:annotation></mml:semantics> </mml:math> = <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>P</mml:mi> <mml:mrow><mml:mrow><mml:mi>M</mml:mi> <mml:mo>,</mml:mo> <mml:mi>C</mml:mi></mml:mrow> <mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> </mml:mrow> </mml:msub> <mml:annotation>${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$</mml:annotation></mml:semantics> </mml:math> ×Δ[CO₂]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhances <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:msub><mml:mi>P</mml:mi> <mml:mrow><mml:mrow><mml:mi>M</mml:mi> <mml:mo>,</mml:mo> <mml:mi>C</mml:mi></mml:mrow> <mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> </mml:mrow> </mml:msub> <mml:annotation>${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$</mml:annotation></mml:semantics> </mml:math> , and (2) intracellular and (3) extracellular carbonic anhydrases (CAs) enhance (Δ[CO₂]) by consuming accumulated or replenishing lost CO₂. We now examine interactions among #1-#3. We assess CO₂ fluxes - produced by addition/removal of extracellular CO₂/HCO₃ ^- - 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 CO₂ fluxes are synergistic among hAQP5, hCA II and bCA, any of which can become rate-limiting, depending on the status of the other two. CO₂/HCO₃ ^- 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 CO₂ channels.