Membrane currents elicited by the epileptogenic drug pentylenetetrazole in the native oocyte ofXenopus laevis

DIDS (4,4''-diisothiocyanato-stilbene-2,2''-disulfonic acid) is a commonly used blocker of plasma membrane anion channels and transporters. We observed that DIDS undergoes decomposition while stored in DMSO (dimethyl sulfoxide) forming a biologically active compound. One decomposition product, called IADS, was identified and synthesized. Voltage-clamp and patch clamp experiments on Xenopus laevis oocytes and human erythrocytes revealed that IADS is able to activate a plasma membrane cation conductance in both cell types. Furthermore, we found that IADS induces hemolysis in red blood cells of healthy donors but fails to hemolyze erythrocytes of donors with cystic fibrosis. Thus, IADS stimulated activation of a cation conductance could form the basis for a novel diagnostic test of cystic fibrosis.

Section: Xenopus Laevis Oocytesmentioning

confidence: 99%

IADS, a Decomposition Product of DIDS Activates a Cation Conductance in <i>Xenopus</i> Oocytes and Human Erythrocytes: New Compound for the Diagnosis of Cystic Fibrosis

Stumpf

Almaça

Kunzelmann

et al. 2006

“…Experiments were performed in Xenopus laevis oocytes, stage VI, obtained as described [9] and stored in Barth medium (87 mM NaCl, 1 mM KCl, 1.5 mM CaCl 2 , 0.8 mM MgSO 4 , 2.4 mM NaHCO 3 , 5 mM HEPES, pH 7.4, containing 100 IU/ml penicilline and 100 µg/ml streptomycine).…”

Section: Oocyte Preparationmentioning

confidence: 99%

Imaging CFTR: A Tail to Tail Dimer with a Central Pore

Schillers

Shahin

Albermann

et al. 2004

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein that belongs to the superfamily of ATP binding cassette (ABC) transporters. Mutations in the CFTR gene cause cystic fibrosis, an autosomal recessive disorder. The function of CFTR is versatile. It can serve as a regulatory protein, as a membrane transporter and as an ion channel. Dimerization of CFTR is necessary for full ion channel function although structural details of CFTR in native membrane are yet unknown. In order to identify CFTR in native plasma membrane we applied atomic force microscopy (AFM) to inside-out oriented membrane patches of CFTR-expressingXenopus laevis oocytes after cAMP stimulation. First, oocytes were injected with CFTR-cRNA and, three days later, voltage-clamped verifying successful CFTR expression and incorporation into the plasma membrane. Then, plasma membrane patches were isolated, placed inside-out on appropriate substrate and incubated with gold-labelled antibodies against the C-terminus of CFTR. Finally, the intracellular surface of the plasma membrane was scanned by AFM. In close vicinity to the immunogold labels we detected ring-like structures with bipartite symmetry. The substructure of the ring, formed by the extramembrane protein domains of CFTR, is consistent with the model of a CFTR dimer. Derived from AFM molecular mass analysis of the intramolecular domains we conclude that two CFTR molecules line up in parallel, tail by tail, forming a pore in its center. This molecular arrangement could represent the CFTR chloride channel configuration, operative in native plasma membrane.

“…The experiments were performed on X. laevis oocytes stage V, which were obtained as described [13] and stored in Barth medium (87 mM NaCl, 1 mM KCl, 1.5 mM CaCl 2 , 0.8 mM MgSO 4 , 2.4 mM NaHCO 3 , 5 mM HEPES, pH 7.4 containing 100 IU/ml penicilline and 100 µg/ml streptomycine). The oocytes were mechanically defolliculated 1 h after collagenase treatment (1 mg/ ml, type D collagenase, Boehringer, Mannheim) and then incubated for 2 h in Barth medium.…”

Section: Oocyte Preparationmentioning

confidence: 99%

Plasma Membrane Plasticity of Xenopus laevis Oocyte Imaged with Atomic Force Microscopy

Schillers

Danker²,

Schnittler³

et al. 2000

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm²) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level.