RESUMO
Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.
Assuntos
Bioquímica/métodos , Lipossomos , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , DNA/metabolismo , Difusão , Eletroquímica , Eletroporação , Fluoresceínas/metabolismo , Fluorescência , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas , Microeletrodos , Microscopia Confocal , Microscopia de Fluorescência , Miniaturização , Técnicas de Patch-Clamp , FosfolipídeosRESUMO
We describe a capillary electrophoresis-patch clamp (CE-PC) analysis of biomolecules that activate ligand-gated ion channels. CE-PC offers a powerful means for identifying receptor ligands based on the combination of the characteristic receptor responses they evoke and their differential electrophoretic migration rates. Corner frequencies, membrane reversal potentials, and mean and unitary single-channel receptor responses were calculated from currents recorded with patch clamp detection. This information was then combined with the electrophoretic mobility of the receptor ligand, which is proportional to the charge-to-frictional-drag ratio of that species. We applied CE-PC to separate and detect the endogenous receptor agonists gamma-aminobutyrate and L-glutamate and the synthetic glutamate receptor agonists N-methyl-D-aspartate and kainic acid. We present dose-response data for electrophoretically separated kainic acid and discuss its implications for making the CE-PC detection system quantitative.
Assuntos
Eletroforese Capilar/métodos , Técnicas de Patch-Clamp , Animais , Eletroforese Capilar/instrumentação , Feminino , Interneurônios/química , Interneurônios/metabolismo , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
We report here a rapid evaporation method that produces in high yield giant unilamellar vesicles up to 50 microns in diameter. The vesicles are obtained after only 2 min and can be prepared from different phospholipids, including L-alpha-phosphatidylcholine (lecithin), dipalmitoleoyl L-alpha-phosphatidylcholine, and beta-arachidonoyl gamma-palmitoyl L-alpha-phosphatidylcholine. Vesicles can be produced in distilled water and in Hepes, phosphate, and borate buffers in the pH range of 7.0 to 11.5 with ionic strengths up to 50 mM. The short preparation time allows encapsulation of labile molecular targets or enzymes with high catalytic activities. Cell-sized proteoliposomes have been prepared in which gamma-glutamyltransferase (EC 2.3.2.2) was functionally incorporated into the membrane wall.
Assuntos
Lipossomos/síntese química , Fosfolipídeos/síntese química , Proteolipídeos , Estabilidade de Medicamentos , Indicadores e Reagentes , Lipossomos/química , Conformação Molecular , Fosfolipídeos/química , Relação Estrutura-Atividade , gama-Glutamiltransferase/química , gama-Glutamiltransferase/metabolismoRESUMO
Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.
