FIGURE 4.

Anionic lipids influence conformational transitions involving the nAChR pore. Conformational transitions of the nAChR pore were probed by monitoring the fluorescence of dibucaine-displaceable ethidium binding. The fluorescence emission of ethidium at 590 nm was monitored in the presence of the nAChR reconstituted into the following: trace i, 3:2 PC/PA; trace ii, 3:2 PC/PG; trace iii, 3:2 PC/PI; trace iv, 3:2 PC/PS; and trace v, PC membranes, using 20 nm excitation and emission slit widths. A, at the indicated times, ∼50 nm nAChR, 500 μm Carb, and 500 μm dibucaine were added to a 0.3 μm ethidium solution. The light gray fluorescence traces were recorded using nAChR preincubated with a large excess of α-bungarotoxin (α-Btx; final concentration = 1.0 μm). The fluorescence traces in the presence and absence of α-bungarotoxin are offset slightly to improve clarity. The sharp spikes in each fluorescence emission trace reflect the scattering of light upon insertion of the pipette into the cuvette. EthBr, ethidium bromide; Dib, dibucaine. B, dibucaine-displaceable ethidium fluorescence emission intensity at 590 nm in the presence (+) or absence (−) of Carb. Error bars are the mean ± S.E. for n = 6 experiments, three measurements each from two different reconstitutions at the indicated lipid composition. a.u., absorbance units. C, schematic for the ethidium (Eth) fluorescence measurements. Ethidium fluoresces weakly in solution (left and right) but with greater intensity when bound to the desensitized nAChR pore (middle).