Anyone out there making planar or tip dipping lipid bi-layers? I have been told lipid formulation is 4:1 POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) to POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Alternatively some authors use only DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). Any thoughts on what’s best for reconstituting with ion channels please?
We were successful in making tip bilayers by the method I could illustrate on the example of reconstitution of the amphotericine B channel in bimolecular membrane.
Reconstitution of amphotericin B / cholesterol channels in lipid bilayers: Lipid bilayers containing pure 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Inc.) were formed on the tip of patch pipettes using the tip-dip technique. The bilayers were formed in asymmetric saline condition by the successive transfer of two lipid monolayers upon the tip of the patch pipette. The extracellular solution (bathing the cis-side of the menbrane) contained (in mM ) 125 NaCl, 5 KCl, 1.25 NaH2PO4, and 5 Tris-HCl at pH 7.4, while the intracellular solution (inside the patch pipette) contained 110 KCl, 4 NaCl, 2 NaH2CO3, 0.1 CaCl2, 1 MgCl2, and 2 MOPS at pH 7.4. A 2:1 (mol/mol) mixture of 0.6 mg
amphotericin B and 0.12 mg cholesterol dissolved in a chloroform-methanol (2:1) solvent was sonicated together with 10 mg phospholipid (in hexane) and 10 ml of extracellular solution in order to form liposomes. The emulsion was then carefully transferred (Fig. 2) to the surface (air-fluid interface) of the solution on the
cis-side of the membrane. Incorporation of amphotericin B into the membrane was achieved by dipping the tip of the patch pipette into the emulsion. The resulting single channel current fluctuations were filtered at 5 kHz and recorded on VHS tapes for later analysis. In certain instances, these single channel current fluctuations were subsequently blocked by the addition of 100 mM tetraethylammonium chloride (TEA) to the cis-side of the membrane.
This method is well documented in two Researchgate publications: "
Conference Paper: LARGE AMPHOTERICIN B / CHOLESTEROL ION CHANNELS" and Article: Amphotericin B and Cholesterol in Monolayers and Bilayers.
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