A Ca2+-activated channel from Xenopus laevis oocyte membranes reconstituted into planar bilayers.

Abstract

Plasma membrane fractions from Xenopus laevis oocytes were incorporated into planar lipid bilayers. We show the existence of numerous Ca2+-activated nonspecific channels that are more permeable to anions. These Ca2+ (13,14) trigger membrane depolarization and oocyte and egg activation.To understand the molecular basis of the fertilization potential and its possible association with intracellular calcium changes, we recently described a simple procedure for enriching plasma membranes of Xenopus oocytes (unpublished data). Here, we have incorporated purified plasma membrane fractions of X. laevis oocytes into planar lipid bilayers. The high impedance of the planar bilayer system allows measurements of small conductance fluctuations with improved time resolution. Furthermore, the voltage across the membrane can be clamped to mimic cell plasma membrane potential. Here, the properties of a Ca '-activated nonspecific channel found abundantly in X. Iaevis oocyte membranes are described. This Ca2+-activated channel may be associated directly with the fertilization potential in X. laevis. MATERIALS AND METHODSOocyte Membranes. Ovaries were surgically removed from gravid X. laevis females immersed in water containing 0.15% tricaine (Calbiochem-Behring), and the tissues were placed in ice-cold modified Barth's medium (MBSH medium) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2 4H2O, 0.41 mM CaC12, 10 mM Hepes (pH 7.2), and 10 mg of benzylpenicillin and streptomycin per liter. After several washes in MBSH medium, oocytes (Dumont stage IV-VI, with an average diameter of 1.0-1.4 mm) were manually isolated from ovary fragments by dissection with fine forceps. The oocytes were exposed to Pronase (75 ,g/ml, from Streptomyces griseus; Calbiochem-Behring) for 10 min at 22-24°C to remove adhering follicular cells. The oocytes were allowed to sediment with six changes of MBSH medium. Oocytes were pooled and lysed by three freeze-thaw cycles in a sorbitol/sucrose medium (175 mM D-sorbitol/0.3 M NaCl/75 mM sucrose/30 mM KCl/10 mM MgCl2/5 mM EDTA/1 mM NaHCO3, pH 7.5, with 1 mM phenylmethylsulfonyl fluoride (Sigma) and aprotinin (FBA Pharmaceuticals, New York) at 0.5 units/ ml). As a second method, oocyte extracts in sorbitol/ sucrose medium were prepared by homogenizing ovary fragments (<2-mm fragments) in a Potter-Elvehjem homogenizer with gentle strokes (pestle clearance, >0.5 mm), which ensured breakage mainly of Dumont stage IV-VI oocytes (unpublished data).The oocyte homogenate was centrifuged at 350 x g for 5 min. The postnuclear supernatant was then centrifuged at 1,500 x g for 10 min. The sediment, after two more cycles of centrifugation (1,500 x g for 10 min at 4°C) (fraction P3), was placed in cellulose nitrate tubes, adjusted to density 1.22 with sucrose, overlayered with discontinuous sucrose layers of densities 1.18 and 1.16, and centrifuged with a SW-27 rotor at 85,000 x g for 90 min at 4°C (unpublished data). Fractions were collected from the top in three main bands (I, II, and ...

Specific Inhibition of the Plasmodial Surface Anion Channel by Dantrolene

“…We distinguished these possibilities by expressing a diverse collection of anion channels or transporters on Xenopus oocytes. We tested CFTR, ClC-1, ClC-5, the oocyte's eCaCC, and MOD-1, a serotonin-gated Cl Ϫ channel (24,52,61,47). Transfected oocytes exhibited currents with mean reversal potentials between Ϫ25 and Ϫ30 mV, consistent with selective Cl Ϫ conduction (not shown).…”

Isolation of plasma membrane complexes fromXenopus oocytes

“…Preparation of plasma membrane fractions from unfertilized and fertilized eggs [26,27] and from ovarian oocytes [30,50] of Xenopus has been reported previously. In the work with eggs, nylon mesh filtration was used to remove vitelline envelopes from homogenates, followed by low speed centrifugation (150 x g, 5 min) to collect plasma membrane and cortex fragments as a fluffy layer on top of the yolk platelet pellet [26,27].…”

“…In the work with eggs, nylon mesh filtration was used to remove vitelline envelopes from homogenates, followed by low speed centrifugation (150 x g, 5 min) to collect plasma membrane and cortex fragments as a fluffy layer on top of the yolk platelet pellet [26,27]. The oocyte protocols utilized either manual rinsing of each individual membrane sheet [30] or a series of low speed centrifugations at 350 and 1500 • g, followed by density gradient centrifugation to isolate plasma membrane-cortical granule complexes [50]. During the initial development of our protocol, we encountered great difficulty in separating large plasma membrane sheets from the underlying yolk platelet pellet when we attempted to use a similar low speed centrifugation to collect PMCs, with considerable fragmentation of the PMCs during their resuspension after each spin.…”

Microelectrode study on the ionic mechanisms which contribute to the noradrenaline‐induced depolarization in isolated cells of the rabbit portal vein

“…However, the shift of the reversal potential of the noradrenaline-induced responses to positive potentials when Nal and NaNO3 were used in the microelectrodes indicates that our conclusions are likely to be valid. The order of halide permeability (I-> Br-> Cl-) has also been found in rat lacrimal glands cells (Evans & Marty, 1986), cultured Schwann cells (Gray et al, 1984) and in Xenopus laevis oocyte membranes reconstituted into planar bilayers (Young et al, 1984). Moreover the same sequence is found for the channel opened by glycine and y-aminobutyric acid (GABA) in mouse cultured spinal neurones (Bormann et al, 1987).…”