The transbilayer distribution of phospholipids in disc membranes is a dynamic equilibrium

Abstract

We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll-floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled phospholipid analogues (SL-PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by . 90% and within less than 30 s after SL-PL addition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spinlabel. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bilayer was determined. It decreased with a mean half-time of , 5 min at 25 8C, indicating rapid redistribution of all spin-labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy-consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.Keywords: disc membrane; flip-flop; phospholipid asymmetry; photoreceptor.The outer segments of retinal rod photoreceptor cells comprise an extensive area of folded membranes, which are arranged mainly in a long, closely spaced stack of disc-like saccules. Disc membranes are organized as lipid bilayers with rhodopsin as the major protein. Lipids account for approximately half of the dry weight of the membranes, and the discs consist of 45 mol% phosphatidylcholine (PtdCho), < 42 mol% phosphatidylethanolamine (PtdEtn) and 13.7 mol% phosphatidylserine (PtdSer) [1]. The most striking feature of the disc lipid composition is the unusually high content of long-chain polyunsaturated acids, particularly in PtdEtn and PtdSer, while the fatty acid residues of PtdCho are more saturated [2]. Several lines of evidence suggest that the fluid phospholipid environment of the disc membrane is important for the rod outer segment (ROS) photoreceptor functions, including the stability and functional regenerability of rhodopsin [3] and the influence of the lipid headgroup and acyl chain composition of phospholipids on the equilibrium between the functionally active photointermediate metarhodopsin II and metarhodopsin I [4]. Depletion of phospholipids, arising from essential v3 fatty acid deficiency, leads to loss of visual function with slowly progressing retinal dystrophy [5,6].Recent i...

Light-induced Reorganization of Phospholipids in Rod Disc Membranes

“…The rapid incorporation of analogues into the disc membrane (data not shown) was in agreement with our recent study found in various disc membrane preparations (pH 7.4) at 25°C (18). After insertion of spin-labeled phospholipids into the outer membrane leaflet of discs at 20°C, the time-dependent redistribution of the SL-PL in the membrane was measured using the BSA extraction method (27,22).…”

“…After insertion of spin-labeled phospholipids into the outer membrane leaflet of discs at 20°C, the time-dependent redistribution of the SL-PL in the membrane was measured using the BSA extraction method (27,22). At times indicated in the figures, 50-l aliquots from the labeled membrane suspension were mixed with 25 l of fatty acid-free BSA solution (3% final concentration (w/v)) for 1 min to extract all lipid analogues accessible to BSA (18). The incubation was performed on ice, to reduce transbilayer movement of lipids, thus avoiding redistribution of SL-PL from the inner to the outer leaflet during BSA extraction.…”

“…The distribution of SL-PL was calculated from ESR spectra of the disc membrane pellet and of the supernatants after BSA back-exchange as described previously (18). In each case, a stable equilibrium distribution was reached with a mean half time of less than 4 min.…”

“…The transbilayer distribution of the main phospholipids in disc membranes in the dark is nearly symmetric for PC and PE but asymmetric for PS, favoring the outer (cytoplasmic) leaflet of the membrane (17,18). The electrical asymmetry of the rhodopsin molecule, normal to the membrane plane, has been made responsible for the asymmetry of the lipid distribution strongly supported by studies on disc membranes in the dark (19,20).…”

Stimulus-evoked outer segment changes occur before the hyperpolarization of retinal photoreceptors

“…The preservation of stimulus-evoked ROS movement in low-sodium medium argues against the possibility that an osmotic change tied to a light-induced change in closure of cGMP-gated ion channels underlies the ROS response. However, remaining open possibilities include stimulus-evoked rhodopsin-, transducin-, and/or PDE-dependent permeability changes [25][26][27], compression of disc membranes [28], and the distribution of the phospholipids in the disc membranes [29]. As TRP clearly depends on a structural perturbation of the OS, these possibilities offer possible avenues for investigating the basis of TRP at a subcellular level.…”