Transient Cholesterol Effects on Nicotinic Acetylcholine Receptor Cell-Surface Mobility

Abstract

To what extent do cholesterol-rich lipid platforms modulate the supramolecular organization of the nicotinic acetylcholine receptor (AChR)? To address this question, the dynamics of AChR particles at high density and its cholesterol dependence at the surface of mammalian cells were studied by combining total internal reflection fluorescence microscopy and single-particle tracking. AChR particles tagged with a monovalent ligand, fluorescent α-bungarotoxin (αBTX), exhibited two mobile pools: i) a highly mobile one undergoing simple Brownian motion (16%) and ii) one with restricted motion (∼50%), the rest being relatively immobile (∼44%). Depletion of membrane cholesterol by methyl-α-cyclodextrin increased the fraction of the first pool to 22% and 33% after 15 and 40 min, respectively; the pool undergoing restricted motion diminished from 50% to 44% and 37%, respectively. Monoclonal antibody binding results in AChR crosslinking-internalization after 2 h; here, antibody binding immobilized within minutes ∼20% of the totally mobile AChR. This proportion dramatically increased upon cholesterol depletion, especially during the initial 10 min (83.3%). Thus, antibody crosslinking and cholesterol depletion exhibited a mutually synergistic effect, increasing the average lifetime of cell-surface AChRs∼10 s to ∼20 s. The instantaneous (microscopic) diffusion coefficient D 2–4 of the AChR obtained from the MSD analysis diminished from ∼0.001 µm2 s−1 to ∼0.0001–0.00033 µm2 s−1 upon cholesterol depletion, ∼30% of all particles falling into the stationary mode. Thus, muscle-type AChR exhibits heterogeneous motional regimes at the cell surface, modulated by the combination of intrinsic (its supramolecular organization) and extrinsic (membrane cholesterol content) factors.

Antibody‐induced crosslinking and cholesterol‐sensitive, anomalous diffusion of nicotinic acetylcholine receptors

“…Acute cholesterol depletion/enrichment was performed prior to fluorescent labeling by treating CHO‐K1/A5 cells with 10–15 mM CDx or CDx‐cholesterol (CDx‐Chol) complexes in Medium 1 (‘M1’: 140 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM KCl in 20 mM HEPES buffer, pH 7.4) (Borroni et al ; Almarza et al ). Samples were taken at 20 min from culture dishes incubated at 37°C in the presence or absence of the cholesterol‐modifying chemical.…”

“…The maximum distance for the merging filter in our experiments was determined from the camera pixel size and the distance‐filter criterion (Lu et al ). Briefly, localized molecules that reappeared in consecutive frames were considered as corresponding to the same molecule if the frame‐to‐frame displacement (tracking radius) was within 106 nm, thus allowing the monitoring of molecules with diffusion coefficients of up to 1.33 µm 2 s −1 , that is, conservatively higher than the upper limit for nAChR nanocluster diffusion estimated from TIRF‐SPT experiments in our laboratory (Almarza et al ). Following the above argument, the ‘Max jump distance’ was deduced by considering the maximum distance that a nAChR is allowed to ‘jump’ within the merge time window established above, that is, the maximum distance that, on average, a single‐molecule can travel in 2 frames.…”

Nicotinic acetylcholine receptor–lipid interactions: Mechanistic insight and biological function

“…Lowering cholesterol levels to disrupt lipid rafts leads to altered nAChR clustering and mobility on cell surfaces, rapid internalization, and ultimately enhances function of the surface-retained nAChRs [139,143,144].…”

Antibody-induced crosslinking and cholesterol-sensitive, anomalous diffusion of nicotinic acetylcholine receptors

“…Acute cyclodextrin-mediated cholesterol depletion/enrichment of cultured cells Acute cholesterol depletion/enrichment was performed prior to fluorescent labeling by treating CHO-K1/A5 cells with 10-15 mM CDx or CDx-cholesterol (CDx-Chol) complexes in Medium 1 ("M1": 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 5 mM KCl in 20 mM HEPES buffer, pH 7.4) (Borroni et al 2007;Almarza et al 2014). Samples were taken at 20 min from culture dishes incubated at 37°C in the presence or absence of the cholesterol-modifying chemical.…”

“…Briefly, localized molecules that reappeared in consecutive frames were considered as corresponding to the same molecule if the frame-to-frame displacement (tracking radius) was within 106 nm, thus allowing the monitoring of molecules with diffusion coefficients of up to 1.33 µm 2 s -1 , i.e. conservatively higher than the upper limit for nAChR nanocluster diffusion estimated from TIRF-SPT experiments in our laboratory (Almarza et al 2014). Following the above argument, the "Max jump distance" was deduced by considering the maximum distance that a nAChR is allowed to "jump" within the merge time window established above, i.e.…”