Functional Incorporation of P-Glycoprotein intoXenopusOocyte Plasma Membrane Fails to Elicit a Swelling-Evoked Conductance

Aleu, Jordi; Ivorra, Isabel; Lejarreta, M.; González-Ros, José M.; Morales, Andrés; Ferragut, J.A.

doi:10.1006/bbrc.1997.7150

Cited by 20 publications

(17 citation statements)

References 33 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%

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%

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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“…Oocyte preparation, microinjection and recording Fully grown Xenopus oocytes were isolated and collagenase treated as previously described [1,19]. Cells were kept until use at 15-16°C in a modified Barth's solution (MBS, in mM): NaCl 88; KCl 1; Ca(NO 3 ) 2 0.33; CaCl 2 0.41; MgSO 4 0.82; NaHCO 3 2.40; 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) 10; pH, 7.4, supplemented with 100 IU/ml penicillin and 0.1 mg/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

“…This approach, which allows the functional characterisation of membrane proteins in their natural environment, has been used recently to study different membrane proteins, such as the P-glycoprotein transporter [1], γ-aminobutyric acid, type A (GABA A ) [23, 24] and glutamate receptors [2]. We have extended this technique by showing that nicotinic acetylcholine (ACh) receptors (nAChRs), purified from Torpedo electroplaques and reconstituted in a defined lipid matrix, can also incorporate into the Xenopus oocyte membrane [19].…”

Section: Introductionmentioning

confidence: 99%

Functional incorporation of exogenous proteins into the Xenopus oocyte membrane does not depend on intracellular calcium increase

Gal

Ivorra

Morales

2000

Pflugers Arch - Eur J Physiol

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Fusion of membranes occurs in diverse biological events and, in most cases, Ca2+ greatly augments its rate. The aim of this work was to study the role played by Ca2+ when transplanting exogenous proteins into Xenopus oocyte membranes. Lipid vesicles carrying nicotinic acetylcholine (ACh) receptors (nAChRs) from Torpedo electroplaques were injected into oocytes. The time course of nAChR incorporation was assessed by recording ACh-evoked currents at different times from injection. An incorporation peak was found at 16 h, but responses were maintained for over 48 h. To assess the role played by Ca2+, two groups were considered: control and chelator-loaded oocytes. In the latter group, cells were incubated with 50 microM 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetrakis(acetoxymethyl)ester (BAPTA-AM) or loaded with ca. 5 nmol ethyleneglycol-bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) 2 h before nAChR injection. Both groups responded to ACh, although the current amplitude was smaller in chelator-loaded than in control cells. These results indicate that the slow fusion of lipoproteosome vesicles with the oocyte plasma membrane does not depend on intracellular Ca2+ increase and therefore belongs to the type called "constitutive". This membrane fusion process is thus different from those involved in resealing of disrupted oocyte membranes or in the fusion of cortical granules with the egg membrane.

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“…Protein determinations were done by the Bradford method (Bio-Rad). Determination of Pgp expression was done using the monoclonal antibody (mAb) C-219 (Centocor Inc.) as previously described (30,31), followed by enhanced chemiluminescence (Amersham International) to develop protein bands.…”

Section: Western Blotmentioning

confidence: 99%

Post-transcriptional Regulation of P-Glycoprotein Expression in Cancer Cell Lines

Gómez-Martínez¹,

García-Morales²,

Carrato³

et al. 2007

Molecular Cancer Research

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The present study of inhibitors shows that the histone deacetylase -induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5 ¶ end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5 ¶ end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5 ¶ untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA. (Mol Cancer Res 2007;5(6):641 -53)

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Functional Incorporation of P-Glycoprotein intoXenopusOocyte Plasma Membrane Fails to Elicit a Swelling-Evoked Conductance

Cited by 20 publications

References 33 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

Functional incorporation of exogenous proteins into the Xenopus oocyte membrane does not depend on intracellular calcium increase

Post-transcriptional Regulation of P-Glycoprotein Expression in Cancer Cell Lines

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