Synergistic roles of Aquaporin 5 and Intra- and Extracellular Carbonic Anhydrases in promoting CO2 Diffusion across the Xenopus Oocyte Plasma Membrane

Key PointsO_LIAccording to Ficks law, transmembrane CO2 flux (JCO2) is the product of membrane permeability (PM,CO2) and transmembrane concentration gradient ({Delta}[CO2]): JCO2=PM,CO2{Delta}[CO2]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhances PM,CO2, and (2) intracellular and (3) extracellular carbonic anhydrases (CAs) enhance {Delta}[CO2] by consuming accumulated or replenishing lost CO2. We now examine interactio ns among #1-#3. C_LIO_LIWe assess CO2 fluxes--produced by addition/removal of extracellular CO2/[Formula]--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). C_LIO_LIEnhancing 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. C_LIO_LICO2/[Formula] addition transiently increases pHS ({Delta}pHS), hCA II augments {Delta}pHS ({Delta}{Delta}pHS), and hAQP5 enhances {Delta}{Delta}pHS ({Delta}{Delta}{Delta}pHS)--a novel tool to assess potential CO2 channels. C_LI CO2 diffusion across plasma membranes depends on both membrane CO2 permeability (PM,CO2) and transmembrane CO2 concentration gradient ({Delta}[CO2])--Ficks 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 introducing extracellular 1.5% CO2/10 mM [Formula] and using microelectrodes to measure (1) maximal extracellular-surface pH increase {Delta}pHS, (2) maximal rate of pHS relaxation (dpHS/dt)Max, and (3) maximal rate of intracellular-pH decrease (dpHi/dt)Max. By itself, hCA II minimally increases {Delta}pHS--measured "trans" to added cytosolic CA (CAi)--even at highest doses (100 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 magnitude--meas ured "trans" to added extracellular CA (CAo)--an effect further doubled by hAQP5. Note: pH measureme nts "cis" to added CAs--pHS for bCA, (dpHi/dt)Max for hCA II--are overwhelmed by enzymatical ly-produced/consumed H+, and cannot provide intuitive insight into CO2 fluxes. Our "trans" pH measurements: (1) confirm synergy between CAo and CAi; establish synergy between hAQP5 and both (2) CAo and (3) CAi; and show that enhancement of {Delta}pHS by CAi ({Delta}{Delta}pHS) is a useful tool for assessing CO2 permeability of membrane proteins (e.g., hAQP5).