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

CO ₂ diffusion across plasma membranes depends on both membrane CO ₂ permeability () and the transmembrane CO ₂ concentration gradient (Δ[CO ₂ ]) – Fick's law. Human aquaporin‐5 (hAQP5) enhances CO ₂ diffusion by increasing , whereas carbonic anhydrases (CAs) do so 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 HCO ₃ ⁻ 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 CO ₂ permeability of membrane proteins (e.g. hAQP5). image

Key points

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

• We assess CO ₂ fluxes – produced by addition/removal of extracellular CO ₂ /HCO ₃ ⁻ – 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 /HCO ₃ ⁻ 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.