Preparation of Giant Myelin Vesicles and Proteoliposomes to Register Ionic Channels

Regueiro, Pilar; Monreal, J.; Díaz, R. S.; Sierra, Felipe

doi:10.1046/j.1471-4159.1996.67052146.x

Cited by 6 publications

(2 citation statements)

References 26 publications

(31 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1999). MPP, a predominant integral membrane protein in the mammalian CNS myelin, is essential for rapid and effective propagation of the action potential along the axon (Regueiro et al . 1996).…”

Section: Unaltered Expression Of Other Neural Proteins In Bmi‐exposedmentioning

confidence: 99%

“…neuron-specific protein that serves as an excellent marker for synaptic terminals and is believed to regulate neurotransmitter release through a phosphorylation-dependent interaction with synaptic vesicles (Hosaka et al 1999). MPP, a predominant integral membrane protein in the mammalian CNS myelin, is essential for rapid and effective propagation of the action potential along the axon (Regueiro et al 1996). As illustrated in Fig.…”

Section: Transcription Of Cx43 and Cx32 Genes Was Increased By Bmi Exmentioning

confidence: 99%

See 1 more Smart Citation

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression

Samoilova

Pelletier

et al. 2003

Journal of Neurochemistry

View full text Add to dashboard Cite

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 lM increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 lM). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMIexposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 lM), but not its inactive analog, oleanolic acid (100 lM), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process. Keywords: bicuculline, carbenoxolone, connexins, epilepsy, gap junction. Abbreviations used: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; APV, 2-amino-5-phosphonovaleric acid; BMI, bicuculline methiodide; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CREB, cAMP response element binding protein; Cx, connexin; FRAP, fluorescence recovery after photobleaching; IPSC, inhibitory post-synaptic current; MPP, myelin proteolipid protein; PAD, primary afterdischarge; PPD, pair-pulse depression; Syn, synapsin.

show abstract

Section: Unaltered Expression Of Other Neural Proteins In Bmi‐exposedmentioning

confidence: 99%

Section: Transcription Of Cx43 and Cx32 Genes Was Increased By Bmi Exmentioning

confidence: 99%

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression

Samoilova

Pelletier

et al. 2003

Journal of Neurochemistry

View full text Add to dashboard Cite

show abstract

Reduced Cortical Synaptic Plasticity and GluR1 Expression Associated with Fragile X Mental Retardation Protein Deficiency

Pelletier

Velazquez

et al. 2002

Molecular and Cellular Neuroscience

239

206

View full text Add to dashboard Cite

A Simple Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

et al. 2010

View full text Add to dashboard Cite

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.

show abstract

Preparation of Giant Myelin Vesicles and Proteoliposomes to Register Ionic Channels

Cited by 6 publications

References 26 publications

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression

Reduced Cortical Synaptic Plasticity and GluR1 Expression Associated with Fragile X Mental Retardation Protein Deficiency

A Simple Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

Contact Info

Product

Resources

About