Wang, D.; Moss, F. J.; Boron, W. F.

CO2 diffusion across plasma membranes depends on both membrane CO2 permeability (PM,CO2) and transmembrane CO2 concentration gradient ({Delta}[CO2])-Fick\'s law. Human aquaporin-5 (hAQP5) accelerates CO2 diffusion by increasing PM,CO2, whereas carbonic anhydrases (CAs) accelerate CO2 diffusion by enhancing CO2 consumption/production and thus {Delta}[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 using microelectrodes to measure (1) maximal extracellular-surface pH change {Delta}pHS, (2) maximal rate of pHS relaxation (dpHS/dt)max, and (3) maximal rate of intracellular-pH change (dpHi/dt)Max elicited by introducing extracellular 1.5% CO2/10 mM HCO-. By itself, hCA II minimally increases {Delta}pHS-measured \"trans\" to added cytosolic CA (CAi)-even at highest doses (1000 ng/oocyte). However, hAQP5 alone triples {Delta}pHS, an effect further doubled by increasing hCA II. By itself, bovine erythrocyte CA (bCA) in the extracellular fluid doubles (dpHi/dt)Max-measured \"trans\" to added extracellular CA (CAo)-an effect further doubled by hAQP5. Note: pH measurements \"cis\" to added CA-pHs for bCA, (dpHi/dt)Max for hCA II-are overwhelmed by enzymatically-produced/consumed H+ and cannot provide intuitive insight into CO2 fluxes. Our \"trans\" pH measurements: (1) confirm synergy between CAo and CAi for CO2 fluxes; establish synergy between hAQP5 and (2) CAo and (3) CAi, and show that the CAi-enhanced {Delta}pHS ({Delta}{Delta}pHS) is a useful tool for assessing the CO2 permeability of membrane proteins (e.g., hAQP5).