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Unformatted text preview: PLANAR BILAYER MEMBRANES M A D E FROM PHOSPHOLIPID MONOLAYERS FORM BY A THINNING PROCESS WALTER D. NILES, RICHARD A. LEVIS,AND FREDRIC S. COHEN Rush MedicalCollege,DepartmentofPhysiology,Chicago,Illinois60612 ABSTRACT We investigated the manner in which planar phospholipid membranes form when monolayers are sequentiallyraised.Simultaneouselectricaland opticalrecordingsshowed thatinitiallya thickfilm forms,and the capacitanceofthefilmincreaseswiththe same timecourse as theobservedthinning.Thediameteroffullythinned membranesvariesfrommembranetomembraneand a torus isreadilyobserved.Thefrequency-dependentadmittance ofthe membrane was measured using a wide-bandwidth voltage clamp whose frequency response isessentially independent ofcapacitative load. The membrane capacitance dominates the totaladmittance and the membrane dielectricisnotlossy.Thespecificcapacitanceofmembranesofseveralmixtures was measured. A schematicdiagram oftheformationofthesemembranesispresented. INTRODUCTION There are two basic methods for forming phospholipid bilayer membranes. In the first,lipidisdissolved in a nonvolatile hydrocarbonsolvent.A smallquantityofthis membrane-formingsolutionisappliedtoaholeinasheet, typicallymadeofTeflon,separatingtwoaqueouscompart- ments.The hydrocarbondrainsintoaGibbs-Plateaubor- der(knownasatorus),whichsurroundsthethinfilmthat forms(Muelleretal.,1963).Thisthinfilm,referredtoasa black lipidmembrane (BLM), consistsofaphospholipid bilayerwith a considerable amount ofdissolvedsolvent (Hanaietal.,1965;White, 1972).Inthesecondmethod, bilayers are formed from monolayers as originally describedbyTakagietal.(1965)andmodifiedbyMontal and Mueller (1972).Monolayersspreadattwoair-water interfacesaresequentiallyraisedoveraholethathasbeen pretreatedwithhydrocarbonssuchasVaselineorsqualene (Montal,1974;ReyesandLatorre,1979),andaphospho- lipid bilayer forms where the monolayers meet. These lattermembranes, which we refer toas M M films, are relativelysolvent-free.They are commonly described as formingviathedirectappositionofthetwo monolayers (Montal,1974;White et al.,1976). Here we report simultaneous optical and electrical studies showingthatcontrary tothecommon view,sequen- tial raisingofthemonolayersresultsinitiallyintheforma- tion ofathickhydrocarbon-phospholipid mixturewithin thehole.Onlywhenthehydrocarbondrainsdoesabilayer form. When the membrane has fullythinned, avisually apparent torus surrounds the bilayer. By measuring the area of thinned M M membranes and using a specially constructed wide-bandwidthvoltageclamp,we measured thespecificcapacitanceofthebilayer. METHODS AND MATERIALS Planarmembraneswereformedbysequentiallyraisingmonolayerspasta 50-300-imdiamholeinapartitionseparatingthetwoaqueouscompart- ments.Teflonpartitions,varyinginthicknessfrom 1to5ml,(Dilectrix, Lockport,NY),wereusedwhentheholewaspretreatedwithsqualene.A Saranwrappartition(Dow Chemical,Indianapolis,IN)wasusedwhen theholewas precoated withpetroleumjelly(Vaseline;Cheesebrough- Ponds, Greenwich, CT). The chamber, milled from Teflon, had glassPonds, Greenwich, CT)....
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This document was uploaded on 08/16/2010.
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