Arterial Blood Pressure, Neuronal Excitability, Mineral Metabolism and Cell Volume Regulation Mechanisms Revealed by Xenopus laevis oocytes

Abstract

Xenopus laevis oocytes have been an invaluable tool to discover and explore the molecular mechanisms and characteristics of many proteins, in particular integral membrane proteins. The oocytes were fundamental in many projects designed to identify the cDNA encoding a diversity of membrane proteins including receptors, transporters, channels and pores. In addition to being a powerful tool for cloning, oocytes were later used to experiment with the functional characterization of many of the identified proteins. In this review I present an overview of my personal 30-year experience using Xenopus laevis oocytes and the impact this had on a variety of fields such as arterial blood pressure, neuronal excitability, mineral metabolism and cell volume regulation.

Keywords: Xenopus laevis; calcium sensing; epilepsy; hypertension; membrane proteins; salt transport.

Figure 1

Expression cloning of the NaCl cotransporter, NCC. mRNA extracted from the winter flounder urinary bladder was injected into Xenopus oocytes and three days later the thiazide-sensitive and Cl⁻-dependent ²²Na⁺-uptake was assessed. Once the expression was established, a cDNA from the urinary bladder was constructed and filters containing about 500 clones each were prepared. Pooled bacteria for each filter were used to extract their cDNA to in vitro transcribe cRNA that was used to inject oocytes and search for NCC activity. As expected, the level of expression from the pooled bacteria was higher than from the total mRNA. Then, cloned from the positive filter were studied by groups until the cRNA transcribed from a single individual clone (C7) induced the presence of the thiazide-sensitive and Cl⁻-dependent ²²Na⁺-uptake, which as expected was higher than that observed from the pooled clones. C7 clone cDNA encoded for NCC [13].

Figure 2

The SLC12A family of electroneutral cation chloride cotransporters. Seven members composing the Na⁺-driven and the K⁺-driven branches are shown, but there are two most distant members SLC12A8 and SLC12A9 not shown. The figure depicts the gene name, the cotransporter’s behavior, the human disease produced by inactivating mutations and the phenotype of the knockout mice. The percentages depict the molecular identity among members of the family. ACCPN = agenesis of the corpus callosum polyneuropathy. EIMFS = epilepsy of infancy with migrating focal seizures.