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. Author manuscript; available in PMC: 2008 Aug 31.
Published in final edited form as: Biochemistry. 2006 Apr 4;45(13):4327–4337. doi: 10.1021/bi052281z

Figure 1. Effect of cholesterol levels in DOPC/DOPA membranes on the photoincorporation of [125I]TID into subunits of the nAChR.

Figure 1

Affinity-purified nAChRs were reconstituted into membranes comprised of DOPC, DOPA and increasing amounts of cholesterol. The molar ratio of each lipid mixture was 3:1:[X] respectively. Reconstituted membranes were equilibrated for 1 h with [125I]TID (0.4 μM) in the absence (− lanes) and in the presence (+ lanes) of 400 μM Carb, irradiated at 365 nm for 7 min, and polypeptides resolved by SDS-PAGE. A, corresponding autoradiographs of gels containing the [125I]TID labeling experiments for representative lipid mixtures. The positions of the nAChR subunits and a proteolytic fragment of the γ-subunit (γ') are indicated on the left. B, for each [125I]TID labeling experiment shown above, individual nAChR subunit bands were excised from the dried gel and the amount of [125I]TID photoincorporated into each subunit determined by γ counting (5 min counting time). [125I]TID incorporation into γ' was added to that of the γ-subunit. Shown are bar graphs of the amount of 125I cpm incorporated into each nAChR subunit in the absence or presence of Carb (−/+) and presented as the average of triplicate determinations from a single labeling experiment (error bars indicate the standard error). C, Due to radioactive decay (125I) the total cpm levels vary significantly from sample to sample and therefore as a normalization function, for each [125I]TID labeling experiment, the ratio of the amount of [125I]TID labeling in the γ– and α– subunit in the absence of agonist was calculated. Shown is the relationship between the molar percentage of cholesterol in the membrane and the functionality of the nAChR as measured by [125I]TID labeling (γ/α ratio). The γ/α ratio points (●) are means of three different [125I]TID labeling experiments (error bars indicate standard error, see also supporting information). For comparison, the γ/α ratios for nAChRs fully stabilized in the resting and desensitized states are indicated with a dotted line.