Functional reconstitution of KCl-evoked, Ca2+-dependent acetylcholine release system inXenopus oocytes microinjected with presynaptic plasma membranes and synaptic vesicles

Canals, Josep M.; Ruiz-Avila, Luis; Cantı́, Carles; Solsona, Carles; Marsal, Jordi

doi:10.1002/(sici)1097-4547(19960415)44:2<106::aid-jnr2>3.0.co;2-h

Cited by 12 publications

(11 citation statements)

References 39 publications

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“…As has been shown previously for fish and mammalian membranes (Marsal et al, 1995;Canals et al, 1996;Aleu et al, 1997), the chick cerebellar membrane fragments become integrated into the oocyte plasma membrane in a seamless way (Fig. 1).…”

Section: Discussionsupporting

confidence: 73%

“…Discrete ovary portions were removed from anesthetized frogs (Canals et al, 1996), and stage VNI oocytes (Dumont, 1972) were individually dissected and kept at 15517°C in sterile modified Barth's solution [lo mM HEPES, pH 7.4, 88 mM NaCl, 1 mM KCI, 0.33 mM Ca(NO,),, 0.82 mM MgSO,, 2.4 mM NaHCO,] supplemented with penicillin (100 IU/ml) and streptomycin (0.1 mg/ml). Oocytes were further treated with collagenase (clostridiopeptidase A; EC 3.4.24.3; Sigma, type IA, 0.5 mg/ ml) for 50-60 min at room temperature to remove enveloping cells .…”

Section: Oocyte Preparation and Injectionmentioning

confidence: 99%

“…This method has the advantage of incorporating foreign receptors into the oocyte membrane in their own natural molecular environment, with higher possibilities of their behaving in a thoroughly physiological way (Marsal et al, 1995;Canals et al, 1996).…”

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confidence: 99%

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Guanine Nucleotides, Including GMP, Antagonize Kainate Responses in Xenopus Oocytes Injected with Chick Cerebellar Membranes

Aleu¹,

Barat²,

Solsona³

et al. 1999

Journal of Neurochemistry

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Injection of chick cerebellar membranes, rich in kainate binding sites, into Xenopus oocytes resulted in the structural integration of chick membrane patches into the oocyte plasma membrane that could be easily identified by specific immunofluorescent staining. Application of kainate to the oocyte perfusion medium, under voltage-clamp conditions, induced dose-dependent ( EC, , = 87 +. 14 p M ) inward currents, confirming the functional incorporation to the oocyte of kainate-driven channels. Responses to kainate were consistently nondesensitizing and strongly potentiated by cyclothiazide, suggesting the selective involvement of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-preferring receptors. Binding experiments with (S)-PH]AMPA confirmed the presence in the chick membrane preparation of low-affinity AMPA receptors (K, = 278 nM) amounting to <2% of the total population of kainate binding sites. A tenfold concentration of guanine nucleotides, with different degrees of phosphorylation, blocked the responses to 100 pM kainate by -90%. In the case of GMP, additional concentration-inhibition studies yielded an I C, , of 180 k 11 pM. Our results illustrate the apparent failure of kainate-binding proteins to form functional channels, even when maintaining their own native membrane environment, and confirm the antagonistic behavior of guanine nucleotides, including GMP, toward glutamate receptors, in agreement with previous results of ligand-binding experiments and, more interestingly, with the marked neuroprotective effects of some guanine nucleotides in different excitotoxicity experimental paradigms. Key Words: Chick cerebellum-GMP-Guanine nucleotides-Kainate-Membrane microinjection-xenopus oocytes. J. Neurochem. 72,2170-21 76 (1 999).presumably related to the p-loop involved in the recognition of GTP by G proteins (Saraste et al., 1990), that has been proposed to be part of the glutamate binding site in structural models of ionotropic glutamate receptors (Paas et al., 199627;Sutcliffe et al., 1996). However, the lack of direct involvement of the glycine residues and the variable participation of the critical p-loop lysine residue in GTP binding in glutamate receptors (Paas et al., 1996a,b;Sutcliffe et al., 1998) suggest that the analogy of the latter with other families of GN-binding proteins may be less relevant than anticipated.Although displacement experiments do not give a precise clue as to the antagonistic or agonistic nature of the GNs, further experiments using standard patch-clamp techniques have confirmed the antagonistic behavior of guanosine 5'-0-(2-thiodiphosphate) (GDPPS; a nonhydrolyzable GDP analogue) and GTP toward glutamate, kainate, and NMDA (Budson et al., 1991; Paas et al., 1996a), whereas our own antiexcitotoxic neuroprotection experiments show that GMP and, in most cases, GDPPS do indeed behave as efficient glutamate antagonists, acting at both a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (Ah4PA)kainate and NMDA receptors, with 5'-guanylylimidodiphosphate (GppNHp; a n...

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Section: Discussionsupporting

confidence: 73%

Section: Oocyte Preparation and Injectionmentioning

confidence: 99%

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Guanine Nucleotides, Including GMP, Antagonize Kainate Responses in Xenopus Oocytes Injected with Chick Cerebellar Membranes

Aleu¹,

Barat²,

Solsona³

et al. 1999

Journal of Neurochemistry

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“…Mature female Xenopus laevis were obtained from the Centre d'Elevage des Xénopes, CRBM (Montpellier, France), and kept in chlorine-free fresh water, at 22°C. Discrete ovary portions were removed from anesthetized frogs 23 and stage-V/VI oocytes 24 25 Healthy-looking oocytes from different donors were microinjected with 100 nL of a chick retinal membrane suspension, prepared as just described, by use of an electronic nanoliter injector (model A203XVZ; WPI, Sarasota, FL).…”

Section: Oocyte Preparation and Injectionmentioning

confidence: 99%

“…Agonist-induced currents were recorded using a two-electrode voltage clamp configuration. 23 Intracellular electrodes (0.5-5 M⍀ resistance) were filled with 3 M KCl for voltage recording and with 3 M K ϩ acetate for current injection. The oocyte membrane potential was held at Ϫ70 mV.…”

Section: Electrophysiologymentioning

confidence: 99%

Kainate-Triggered Currents inXenopusOocytes Injected with Chick Retinal Membrane Fragments: Effect of Guanine Nucleotides

Aleu

Barat

Solsona

et al. 2003

Invest. Ophthalmol. Vis. Sci.

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An alternative oocyte microinjection technique to analyze the electrophysiological properties of glutamate receptors in chick retinal membranes is described. The results show the functional activity of putative AMPA-preferring receptors from chick retina and confirm, in the chick retinal model, the antagonistic behavior of guanine nucleotides toward glutamate receptors and their potential role as neuroprotective agents under excitotoxic conditions.

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ATP release from the electric organ of Torpedo and from Xenopus oocytes

Escalada

Aleu

Bodas

et al. 2001

Drug Development Research

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A field-stimulated electric organ generated an electric field and release of a large quantity of ATP. Oocytes injected with mRNA from a Torpedo electric organ released ATP when stimulated with carbamylcholine. We set up a configuration recording that let us simultaneously monitor the oocyte ionic currents and ATP release from a single oocyte. Hyperpolarization pulses applied to Xenopus oocytes induced an inward current and release of ATP independently of the expression of the ClC-0 chloride channel. The current was insensitive to substitution of ions in the extracellular saline solution, either anions or cations. The reversal potential of the current was established between +40 and +100 mV, which is in the same range as the equilibrium potential of ATP -4 . There was a correlation between the magnitude of the inward current and the amount of ATP released. The inward current closed slowly. During this closing period there was still release of ATP, which is sensitive to the holding potential. Positive potentials cut the amount of ATP released. The evoked current and ATP release were both sensitive to inhibitors of E-NTPDase activity, such as suramin, 4-acetamido-4′isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′diisothiocyanatostilbene-2′-disulfonic acid (DIDS), and a high concentration of calcium and gadolinium ions. The expression of CD39 increased the size of the hyperpolarizing induced current. By reverse transcription polymerase chain reaction, we found a protein homologous to CD39. The relationship between the results obtained in the electric organ and oocytes is that, in both cases, stimulation or hyperpolarization causes a change in the intracellular ion concentration, producing an osmotic stress, which, in turn, triggers ATP release. Stimulation or hyperpolarization may alter the intercellular ion concentration, leading to osmotic stress and the release of ATP in both electric organ and oocytes. Drug Dev.

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Functional reconstitution of KCl-evoked, Ca2+-dependent acetylcholine release system inXenopus oocytes microinjected with presynaptic plasma membranes and synaptic vesicles

Cited by 12 publications

References 39 publications

Guanine Nucleotides, Including GMP, Antagonize Kainate Responses in Xenopus Oocytes Injected with Chick Cerebellar Membranes

Guanine Nucleotides, Including GMP, Antagonize Kainate Responses in Xenopus Oocytes Injected with Chick Cerebellar Membranes

Kainate-Triggered Currents inXenopusOocytes Injected with Chick Retinal Membrane Fragments: Effect of Guanine Nucleotides

ATP release from the electric organ of Torpedo and from Xenopus oocytes

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