Synergistic roles of Aquaporin 5 and Intra- and Extracellular Carbonic Anhydrases in promoting CO2Diffusion across the Xenopus Oocyte Plasma Membrane

Abstract Key Points According to Fick’s law, transmembrane CO2 flux (JCO2) is the product of membrane permeability (PM,CO2) and transmembrane concentration gradient (Δ[CO2]): JCO2=PM,CO2Δ[CO2]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhances PM,CO2, and (2) intracellular and (3) extracellular carbonic anhydrases (CAs) enhance Δ[CO2] by consuming accumulated or replenishing lost CO2. We now examine interactio ns among #1–#3. We assess CO2 fluxes—produced by addition/removal of extracellular CO2/—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/ addition transiently increases pHS (ΔpHS), hCA II augments ΔpHS (ΔΔpHS), and hAQP5 enhances ΔΔpHS (ΔΔΔpHS)—a novel tool to assess potential CO2 channels. Key PointsCO2 diffusion across plasma membranes depends on both membrane CO2 permeability (PM,CO2) and transmembrane CO2 concentration gradient (Δ[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 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 and using microelectrodes to measure (1) maximal extracellular-surface pH increase ΔpHS, (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 ΔpHS—measured “trans” to added cytosolic CA (CAi)—even at 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—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 ΔpHS by CAi (ΔΔpHS) is a useful tool for assessing CO2 permeability of membrane proteins (e.g., hAQP5). Competing Interest Statement The authors have declared no competing interest. Footnotes 1) The title has been slightly changed to “Synergistic Roles of Aquaporin 5 and Intra- and Extracellular Carbonic Anhydrases in Helping CO₂ Move through the Xenopus Oocyte Plasma Membrane.” 2) Running heading is now: AQP5 and CA synergism in transmembrane CO₂ diffusion. 3) We have eliminated the Abbreviations section from the previous version. 4) The introduction, abstract, and key points have undergone minor revisions. 5) Methods includes additional description of statistical analyses for Figures 3A-D vs. 8A-D and Figures 6A-D vs. Figure 10A-D 6) The results section underwent significant revisions to present the findings more clearly. Includes new comparisons of measured parameters +/− bovine carbonic anhydrase in the bulk extracellular fluid (bECF) 7) We made moderate revisions to the discussion section to incorporate updated analyses from the results, along with other minor changes for improved clarity. 8) Two additional citations added in references 9) New statistical tables 8E-8H and 10E-10H are available for comparisons of measured parameters, +/− bovine carbonic anhydrase, in the bECF. ↵12 2.22×10−308 is the smallest possible value for double type data that a 64-bit systemis able to distinguish. ↵13 2.22×10−308 is the smallest possible value for double type data that a 64-bit systemis able to distinguish ↵14 2.22×10−308 is the smallest possible value for double type data that a 64-bit systemis able to distinguish