The role of cholesterol in rod outer segment membranes

Abstract

The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evlauated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholestrol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthremore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting to turnover of the protein or protein modification.

Introduction

Cholesterol is an essential membrane constituent of most animal cells. This sterol has been shown to be involved in a wide variety of critical cell functions, including modulation of enzyme function, permeability, fusion and receptor function. Because of its importance to normal membrane function, the mechanism of cholesterol action in biological membranes has been the focus of intensive investigation for decades. Not surprisingly, it is now clear that cholesterol exerts complex, multifaceted effects on cellular membranes. The critical importance of cholesterol to normal functioning of cell membranes may lay in its ability to both alter fundamental properties of the phospholipid bilayer and to interact directly with specific membrane proteins. This duel role of cholesterol was described in a review by Yeagle in 1991 [1]. In this review, the complex effects of cholesterol were used to explain the correlation between optimal protein function and the normal cholesterol content of cells. One notable example is for the kidney Na⁺/K⁺ ATPase, which exhibits maximal activity when it is in a membrane containing the physiological levels of cholesterol. Studies since that time on the oxytocin and cholescytokinin receptors have further supported this duel role of cholesterol in membrane function [2]. That work indicated that the cholescyskinin receptor is modulated through the bulk lipid properties while the oxytocin receptor is modulated both through the bulk lipid and by direct cholesterol interactions. This was also further supported by modeling studies of cholesterol interaction with the cholescytokinin and oxytocin receptors [3]. Recently, direct cholesterol protein binding has been implicated in the mechanism for controlling membrane cholesterol composition [4].

The effects of cholesterol have been extensively investigated in many simple phospholipid bilayer systems. In these systems, it was demonstrated that cholesterol alters the properties of the bulk bilayer phase by interacting with the phospholipids. These lipid bilayer studies demonstrated that cholesterol increases the bilayer thickness [5] and decreases the membrane permeability both to small uncharged polar molecular species and to ions. This has been the subject of several reviews [6], [7], [8], [9], [10]. The effect of cholesterol on ion leakage is particularly intriguing. In animal cells the plasma membrane maintains an essential electrochemical gradient across the membrane through the pumping action of Na⁺/K⁺ ATPases. Therefore leakage of ions across the membrane entails a cost of metabolic energy in the form of ATP. Na⁺ leakage across a bilayer that contains both phospholipids and proteins can be at least partially overcome if cholesterol is present in the membrane [11]. It was proposed that the importance of cholesterol in the plasma membrane may be to reduce the dissipation of the Na⁺ gradient and thus conserve metabolic energy [9].

Many years ago, it was also observed that cholesterol broadens the gel to liquid crystal phase transition temperature of phospholipid bilayers as reviewed by Yeagle [7], [12]. Cholesterol does this by disordering the packing of the phospholipid hydrocarbon acyl chains below the transition, but increasing the ordered packing of these hydrocarbons above the transition. This observation led to the hypothesis that cholesterol modulates the lipid gel to liquid crystalline phase transition in biological membranes. Since that time a role of cholesterol has been proposed to be that of a primary modulator of the “fluidity” of the hydrocarbon region of the bilayer. While “fluidity is a poor term to describe membrane dynamics, in this context “fluidity” generally refers to the motional freedom and packing of the hydrocarbon side chains. In this context, cholesterol was acknowledged to be important in modulating the dynamics of the hydrocarbon region of the bilayer. As described later in this review, cholesterol is able to modulate membrane protein function through its impact on lipid packing.

No other sterol can completely substitute for cholesterol in mammalian cell membranes. That is, generally mammalian cells are not viable when totally depleted of cholesterol. This is likely because some cholesterol effects are due to unique structural features of cholesterol. However, other sterols may approximate certain of the cholesterol membrane effects and substitute for cholesterol in that particular function. Thus if the cell membrane cholesterol is depleted, but not eliminated, addition of another sterol can substitute for the bulk membrane cholesterol and produce a similar effect on membrane function as original level of cholesterol alone [13]. This is also consistent with a multifunctional role for cholesterol.

Rod photoreceptor cells provide a unique system in which to study the synthesis, distribution and function of cholesterol within a single differentiated cell. The synthesis of cholesterol, the cellular distribution of cholesterol and the effect of membrane cholesterol on various functional parameters have been investigated in this cell using a range of techniques. Therefore, this system provides an ideal example of cellular membranes in which the in vivo role of cholesterol can be correlated with biophysical studies, in particular, the effect of cholesterol on rhodopsin, the photoreceptor and archetype for the family of G-protein receptors. This is particularly relevant because cholesterol has been implicated in the regulation of other G-protein receptors (review [14]). These include the nicotinic acetylcholine [15], [16], cholescytokinin [2], oxytocin [17], [18], [2], [19] serotonin [20] and transferrin [21] receptors. Therefore, an examination of the role of cholesterol on rhodopsin is likely to provide insight with respect to other G-protein receptors.