New model system for the heterologous expression and functional evaluation of ammonia (NH3/NH4 +) transporters using Rana catesbeiana oocytes: an alternative to the Xenopus laevis system

Abstract

Gas transport across cell membranes is critical in both physiology and pathophysiology. Currently, several lines of experimental arguments challenge the classical dogma that all gases pass through all membranes merely by dissolving in the lipid phase of the membrane. The first evidences challenging this dogma came from Dr Walter F. Boron laboratory, which demonstrated that 1) apical membranes of gastric-gland cells and apical membranes of colonic crypts are impermeable to CO2 and NH3 and 2) the gas CO2 can pass through a water channel (Aquaporin 1, AQP1) expressed in oocytes of Xenopus laevis, providing the first evidence of a gas (CO2) passing by a channel (AQP1). However, gas channels could only be effective if 1) the "background" permeability of the membrane to the gas is lower (small) compared to the contribution of the channels, 2) when the gas concentration gradient for driving diffusion is low, and 3) when the physiologocial demand for gas flux is high. Xenopus oocyte is commonly used for heterologous expression of membrane protein-channels and transporters-valuable to study the electrophysiology and molecular biology of new proteins in vivo. However, because of bureaucratic delays in importing the Xenopus laevis frogs to begin the implementation of the Xenopus oocyte model in our Department, it became necessary to seek and develop an alternative heterologous expression model, using oocytes from frogs that are commercialized in Brazil, such as the oocytes from the frog specie-Rana catesbeiana. Thus, we have been using Rana oocytes as an oocyte-expression system to study the mechanisms of NH3 transport through proteins: AQPs 3, 7, 8, and 9, glycosylated Rh proteins RhAG, RhBG, and RhCG, as well the urea transporter UT-B and UT-A1. These proteins are expressed in tissues involved in NH3/NH4+ homeostasis or transport.The oocytes NH3 permeability will be analyzed using microelectrodes to simultaneously monitor membrane potential (Vm), intracellular pH (pHi) and surface pH (pHS) caused by the influx of dissolved gases (CO2 or NH3) or ions between extra and intracellular space of Rana oocytes after exposure of specific solutions. The Principle: if NH3 enters the cell, the NH3 influx causes a transient fall in pHS, reducing the [NH3] at the extracellular surface of the oocyte. The depleted [NH3] at the cell surface is replenished by diffusion from the bulk extracellular fluid and from the reaction NH3 + H+ ® NH4+ at the cell surface. The newly produced H+ originating from this reaction causes a detectable shift in pHS, which is proportional to the NH3 influx. The pHS fall is measured with the surface pH electrode. The proposed work is part a project designed to understand how NH3 move through channels and, at a more integrated level, the physiological importance of gas channels. In the future, our research could make part of other studies that are reorganizing our thinking of how gases cross cell membranes. (AU)