Resolution of complex fluorescence spectra of lipids and nicotinic acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid microenvironment

Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Förster resonance energy transfer (FRET) was observed between the protein and pyrene-PC and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to fluorescence spectra of pyrene-PC in liposomes with different DOPC/DOPA ratios, generating a model that was tested by an internal validation (leave-one-out cross-validation) and was further used to predict the apparent lipid molar ratio in AChR-containing samples. The values predicted for DOPA, the lipid with the highest Tm, indicate that the protein exerts a rigidifying effect on its lipid microenvironment. A similar conclusion was reached from excimer formation of pyrene-PC, a collisional-dependent phenomenon. The excimer/monomer ratio (E/M) at different DOPC/DOPA molar ratios revealed the restricted diffusion of the probe in AChR-containing samples in comparison to pure lipid samples devoid of protein. FRET from the AChR (donor) to pyrene-PC (acceptor) as a function of temperature was found to increase with increasing temperature, suggesting a shorter distance between AChR and pyrene PC. Taken together, the results obtained by MA on complex spectra indicate that the AChR rigidifies its surrounding lipid and prefers DOPA rather than DOPC in its immediate microenvironment

Agonist-Induced Hump Current Production In Heterologously-Expressed Human α4β2-Nicotinic Acetylcholine Receptors

Aim: To characterize the functional and pharmacological features of agonist-induced hump currents in human α4β2-nicotinic acetylcholine receptors (nAChR). Methods: Whole-cell and outside-out patch recordings were performed using human α4β2-nAChR heterologously expressed in stably-transfected, native nAChR-null subclonal human epithelial 1 (SH-EP1) cells. RT-PCR was used to test the mRNA expression of transfected nAChR. Homology modeling and ace-tylcholine (ACh) docking were applied to show the possible ACh-binding site in the channel pore. Results: The rapid exposure of 10 mmol/L ACh induced an inward current with a decline from peak to steady-state. However, after the removal of ACh, an additional inward current, called \ hump\ current, reoccurred. The ability of agonists to produce these hump currents cannot be easily explained based on drug size, charge, acute potency, or actions as full or partial agonists. Hump currents were associated with a rebound increase in whole-cell conductance, and they had voltage dependence-like peak currents induced by agonist action. Hump currents blocked by the α4β2-nAChR antagonist dihydro-β-erythroidine were reduced when α4β2-nAChR were desensitized, and were more pronounced in the absence of external Ca2+. Outside-out single-channel recordings demonstrated that compared to 1 μmol/L nicotine, 100 μmol/L nicotine reduced channel current amplitude, shortened the channel mean open time, and prolonged the channel mean closed time, supporting an agonist-induced open-channel block before hump current production. A docking model also simulated the agonist-binding site in the channel pore. Conclusion: These results support the hypothesis that hump currents reflect a rapid release of agonists from the α4β2-nAChR channel pore and a rapid recovery from desensitized α4β2- nAChR. © 2008 CPS and SIMM