Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labele d phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) anal og (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by compari son to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/c holesterol of molar ratio 1, that DRM contains a substantial amount of liqu id ordered phase: 1)The rotational diffusion rates (R-perpendicular to) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as th ose in molar ratio DPPC/Chol = 1 dispersions, and they are substantially gr eater than R-perpendicular to in pure DPPC dispersions in the gel phase stu died above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temp eratures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispers ions, but are greater than those in DPPC dispersions in both the gel and li quid crystalline phases. 3) Similarly, R-perpendicular to for 5PC and CSL i n DRM is greater than in pure SM dispersions in the gel phase, and S for th ese labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R-perpendicular to of SD-Tempo in DRM is gre ater than in dispersions of SM in both gel and liquid phases, consistent wi th the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases tin absolute values), indicating that th e headgroup region in DRMs is less ordered than in pure SM. These results s upport the hypothesis that plasma membranes contain DRM domains with a liqu id ordered phase that may coexist with a liquid crystalline phase. There al so appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes c ontaining high concentrations of cholesterol also contain a liquid ordered phase.