Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Methods (Impact Factor: 3.65). 05/2010; 51(1):134-45. DOI: 10.1016/j.ymeth.2009.12.012
Source: PubMed


The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (approximately 1mm diameter), (b) it has an established capacity to produce-from microinjected mRNAs or cRNAs-exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion transporters as green fluorescent protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl(-), H(+) (and hence base equivalents like OH(-) and HCO(3)(-)), K(+), and Na(+) is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates.

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    • "In fact, X. laevis oocytes are able to efficiently translate exogenous mRNA into proteins upon injection of the corresponding mRNA. More particularly, the use of X. laevis oocytes has been extremely fruitful in the expression of receptor, channel, and transporter proteins, to subsequently take advantage of sensitive techniques such as electrophysiology and radiotracer uptake as extensively reported in the literature (Cucu et al., 2004; Mari et al., 2006; Musa-Aziz et al., 2010). Compared with those techniques, the advantage of AFM lies in its ability to obtain nanometer-scale and time-lapse information on the submolecular structure and supramolecular assembly of functional membrane proteins, for example, visualizing conformational changes (Scheuring et al., 2006; Mari et al., 2011; Picas et al., 2013). "
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    ABSTRACT: Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.
    Journal of Molecular Recognition 11/2014; 27(11). DOI:10.1002/jmr.2390 · 2.15 Impact Factor
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    • "Intact Xenopus oocytes have also been key for membrane channel and receptor studies, including the first electrophysiological analysis of cloned membrane channels and receptors (Kusano et al., 1977). Xenopus oocytes enable rapid assays of channel and transporter protein activities because they correctly process the proteins, insert them into the cell membrane, and can be cultured for days (Musa-Aziz et al., 2010; Sobczak et al., 2010). This experimental approach has led to important discoveries in the membrane channels, receptors , and transporters of nervous, cardiac, auditory, and nephric systems. "
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    ABSTRACT: The frog Xenopus has been vital for biomedical science for over 80 years, contributing to diverse fields from cell signaling, cell and developmental biology, to ion channel physiology and toxicology. Its experimentally manipulable oocytes and embryos provide abundant material for molecular and biochemical approaches for a wide range of gene discovery and protein function studies. In recent years, the Xenopus community has invested in key resources for functional genomics, including genome-wide full-length cDNA collections and genome assemblies as well as genetic tools. These assets combine with Xenopus' extensive range of functional assays to create exciting new research avenues with medical as well as basic applications. This review describes how these resources were developed and what new tools are on the horizon.
    genesis 03/2012; 50(3):133-42. DOI:10.1002/dvg.22015 · 2.02 Impact Factor
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    • "Measurements of membrane conductance (using voltage-clamp circuitry), Na + activity and pH i (using ion-selective microelectrodes) were performed as recently described in detail (Toye et al. 2006; Musa-Aziz et al. 2010). These methods are summarized below. "
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    ABSTRACT: The renal electrogenic Na(+)/HCO(3)(−) cotransporter (NBCe1-A) contributes to the basolateral step of transepithelial HCO(3)(−) reabsorption in proximal tubule epithelia, contributing to the buffering of blood pH. Elsewhere in the body (e.g. muscle cells) NBCe1 variants contribute to, amongst other processes, maintenance of intracellular pH. Others have described a homozygous mutation in NBCe1 (NBCe1-A p.Ala799Val) in an individual with severe proximal renal tubular acidosis (pRTA; usually associated with defective HCO(3)(−) reabsorption in proximal tubule cells) and hypokalaemic periodic paralysis (hypoPP; usually associated with leaky cation channels in muscle cells). Using biotinylation and two-electrode voltage-clamp on Xenopus oocytes expressing NBCe1, we demonstrate that the mutant NBCe1-A (A(A799V)) exhibits a per-molecule transport defect that probably contributes towards the observed pRTA. Furthermore, we find that A(A799V) expression is associated with an unusual HCO(3)(−)-independent conductance that, if associated with mutant NBCe1 in muscle cells, could contribute towards the appearance of hypokalaemic paralysis in the affected individual. We also study three novel lab mutants of NBCe1-A: p.Ala799Ile, p.Ala799Gly and p.Ala799Ser. All three exhibit a per-molecule transport defect, but only A(A799I) exhibits an A(A799V)-like ion conductance. A(A799G) and A(A799S) exhibit unusual outward rectification in their HCO(3)(−)-dependent conductance and A(A799G) exhibits reduced sensitivity to both DIDS and tenidap. A799G is the first mutation shown to affect the apparent tenidap affinity of NBCe1. Finally we show that A(A799V) and A(A799I), which accumulate poorly in the plasma membrane of oocytes, exhibit signs of abnormal intracellular accumulation in a non-polarized renal cell-line.
    The Journal of Physiology 02/2012; 590(Pt 8):2009-34. DOI:10.1113/jphysiol.2011.224733 · 5.04 Impact Factor
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