Integration and recording of a reconstituted voltage-gated sodium channel in planar lipid bilayers.
Functional assays for membrane proteins become increasingly important in biosciences. We demonstrate the integration of reconstituted bacterial voltage-gated sodium channels (NaChBac) into preformed free-standing lipid bilayers by using the nystatin-ergosterol method to promote proteoliposome fusion. Vesicle delivery and subsequent NaChBac activity were monitored, the orientation of the transferred ion channels was assessed measuring at both, positive and negative holding potentials and the channel specificity was demonstrated by adding the blocker nimodipine. A conductance of 120 pS per channel and an opening time in the range of seconds have been observed. Interestingly, we found that fusion of proteoliposomes into preformed free-standing bilayers is limited, if hydrophobically silanized silicon nitride membranes are used as the supporting material. In this case the diameter of the liposome had to be at least 20 times smaller compared to that of the pore to render fusion possible.
Studer
A
A
Paul Scherrer Institut, Department of Biology and Chemistry, Biomolecular Research Laboratory, CH-5232 Villigen PSI, Switzerland. a.studer@imperial.ac.uk
Demarche
S
S
Langenegger
D
D
Tiefenauer
L
L
D001426:Bacterial Proteins / Q000737:chemistry*; D040761:Biomimetic Materials; D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry*; D008564:Membrane Potentials; D015222:Sodium Channels / Q000737:chemistry*; D018511:Systems Integration
D001426:Bacterial Proteins; D008051:Lipid Bilayers; C443666:NaChBac protein, bacteria; D015222:Sodium Channels