Synergistic roles of aquaporin 5 and intra- and extracellular carbonic anhydrases in promoting CO2 diffusion across the Xenopus oocyte plasma membrane.

CO2 diffusion across plasma membranes depends on both membrane CO2 permeability ( P M , C O 2 ${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ) and the transmembrane CO2 concentration gradient (Δ[CO2]) - Fick's law. Human aquaporin-5 (hAQP5) enhances CO2 diffusion by increasing P M , C O 2 ${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ , 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 (ΔpHS), (2) the maximal rate of pHS relaxation (dpHS/dt)Max and (3) the maximal rate of intracellular pH decrease (dpHi/dt)Max. By itself hCA II minimally increases ΔpHS - measured on the side of the membrane opposite to the added cytosolic CA (CAi) - even at our highest doses (100 ng/oocyte). However hAQP5 alone triples ΔpHS, an effect further doubled by increasing hCA II. By itself bovine erythrocyte CA (bCA) in the extracellular fluid doubles (dpHi/dt)Max magnitude - measured on the side of the membrane opposite to the added extracellular CA (CAo) - an effect further doubled by hAQP5. Our pH measurements (1) confirm synergy between CAo and CAi; establish synergy between hAQP5 and both (2) CAo and (3) CAi; and (4) show that the ability of CAi to enhance ΔpHS 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 ( J C O 2 ${J_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ) is the product of membrane permeability ( P M , C O 2 ${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ) and transmembrane concentration gradient (Δ[CO2]): J C O 2 ${J_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$  =  P M , C O 2 ${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ×Δ[CO2]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhances P M , C O 2 ${P_{{\mathrm{M,C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ , 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 (pHS) and intracellular pH (pHi) 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 pHS (ΔpHS), hCA II augments ΔpHS (ΔΔpHS) and hAQP5 enhances ΔΔpHS (ΔΔΔpHS) - a novel tool to assess potential CO2 channels.