Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes

摘要

Human carbonic anhydrase IV (CA IV) is GPI-anchored to the outer membrane surface, catalyzing CO2/HCO3⁻ hydration-dehydration. We examined effects of heterologously expressed CA IV on intracellular-pH (pHi) and surface-pH (pHS) transients caused by exposing oocytes to CO2/HCO3⁻/pH 7.50. CO2 influx causes a sustained pHi fall and a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA IV increases magnitudes of maximal rate of pHi change (dpHi/dt)max, and maximal pHS change (ΔpHS) and decreases time constants for pH_i changes (τ_pHi) and pH_S relaxations (τ_pHS). Decreases in time constants indicate that CA IV enhances CO₂ fluxes. Extracellular acetazolamide blocks all CA IV effects, but not those of injected CA II. Injected acetazolamide partially reduces CA IV effects. Thus, extracellular CA is required for, and the equivalent of cytosol-accessible CA augments, the effects of CA IV. Increasing the concentration of the extracellular non-CO₂/HCO₃⁻ buffer (i.e., HEPES), in the presence of extracellular CA or at high [CO₂], accelerates CO₂ influx. Simultaneous measurements with two pH_S electrodes, one on the oocyte meridian perpendicular to the axis of flow and one downstream from the direction of extracellular-solution flow, reveal that the downstream electrode has a larger (i.e., slower) τ_pHS, indicating [CO₂] asymmetry over the oocyte surface. A reaction-diffusion mathematical model (third paper in series) accounts for the above general features, and supports the conclusion that extracellular CA, which replenishes entering CO₂ or consumes exiting CO₂ at the extracellular surface, enhances the gradient driving CO₂ influx across the cell membrane.