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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Mar 24;177(11):2456–2465. doi: 10.1111/bph.15027

Functional marriage in plasma membrane: Critical cholesterol level–optimal protein activity

Ulises Meza 1,✉, Mayra Delgado‐Ramírez 1, Catalina Romero‐Méndez 1, Sergio Sánchez‐Armass 1, Aldo A Rodríguez‐Menchaca 1,✉
PMCID: PMC7205822  PMID: 32060896

Abstract

In physiology, homeostasis refers to the condition where a system exhibits an optimum functional level. In contrast, any variation from this optimum is considered as a dysfunctional or pathological state. In this review, we address the proposal that a critical cholesterol level in the plasma membrane is required for the proper functioning of transmembrane proteins. Thus, membrane cholesterol depletion or enrichment produces a loss or gain of direct cholesterol–protein interaction and/or changes in the physical properties of the plasma membrane, which affect the basal or optimum activity of transmembrane proteins. Whether or not this functional switching is a generalized mechanism exhibited for all transmembrane proteins, or if it works just for an exclusive group of them, is an open question and an attractive subject to explore at a basic, pharmacological and clinical level.

Abbreviations

ATP

adenosine triphosphate

CARC

reverse version of the CRAC

CRAC

cholesterol recognition amino acid consensus

Kir

inwardly rectifying K+ channel

KV

voltage‐dependent K+ channel

NCX

Na+/Ca2+ exchanger

Rhodo

rhodopsin

1. INTRODUCTION

Plasma membrane delimits the interior and exterior of the cells. During the biological evolution, its development represented a critical event in the process of the origin of the first cells (Monnard & Deamer, 2002). The plasma membrane is a dynamic boundary structure mainly constituted by lipids, proteins and carbohydrates. The structural co‐existence and functional co‐ordination of these elements are essential to maintain an optimal (or basal) cell activity level (cell homeostasis). Therefore, it should be expected that any significant variation in both the concentration of these components and their functions results in a dysfunctional or pathological condition.

https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2718 is the most abundant lipid in the plasma membrane of animal cells (Lambropoulos, Garcia, & Clarke, 2016). Depending on the cell type, it represents the ~30–40 mol% of the total lipids of this structure (Ikonen, 2008; Ikonen, 2018). It is known that cholesterol influences the biophysical properties of the plasma membrane by promoting the liquid ordered (Lo) phase separation from the membrane bulk or liquid disordered phase (Ld; Ipsen, Karlström, Mouritsen, Wennerström, & Zuckermann, 1987). Actually, a functional compartmentalization is proposed to explain the efficiency of signal transduction at the low physiological surface concentrations of the signalling partners by their grouping inside specialized rich cholesterol nanodomains or rafts (Pike, 2006; Simons & Ikonen, 1997). It has been estimated that lipid rafts (Lo phase) cover around 10% of the total plasma membrane area (Meyer et al., 2006; Kusumi et al., 2012).

Variations from cholesterol basal level induce changes in the expression, distribution and function of different transmembrane proteins like https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=694, ion channels and https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=691 (Vemuri & Philipson, 1988; Sotomayor, Aguilar, Cuevas, Helms, & Jameson, 2000; Sooksawate & Simmonds, 2001a; Harikumar et al., 2005; Barrantes, 2007; Picazo‐Juárez et al., 2011; Levitan, Singh, & Rosenhouse‐Dantsker, 2014; Licon et al., 2015; Desai, Dong, & Miller, 2016; Lange & Steck, 2016; Delgado‐Ramírez, Sánchez‐Armass, Meza, & Rodríguez‐Menchaca, 2018; Garcia et al., 2019; McGraw, Yang, Levental, Lyman, & Robinson, 2019).

In general, two mechanisms are proposed to explain the functional effects of cholesterol on transmembrane proteins (Figure 1):‐ 1. direct interaction of cholesterol–protein, through specific cholesterol‐binding domains [e.g. cholesterol recognition amino acid consensus (CRAC)] and 2. the reverse version of the CRAC referred to as CARC domain; Fantini & Barrantes, 2013; Fantini, Di Scala, Evans, Williamson, & Barrantes, 2016] and induction of changes in the biophysics properties of the plasma membrane (Khatibzadeh, Gupta, Farrell, Brownell, & Anvari, 2012; Lundbæck & Andersen, 2012; Delgado‐Ramírez et al., 2018). Diverse pharmacological, biophysical and molecular approaches, including docking and molecular dynamic simulations are commonly used to discriminate between these two possibilities (Khatibzadeh et al., 2012; Fantini et al., 2016; Delgado‐Ramírez et al., 2018). Particularly, application of methyl‐β‐cyclodextrin or methyl‐β‐cyclodextrin:cholesterol complex represents a standard approach utilized to deplete (~30–80%) or load (~100–300%) cholesterol in the plasma membrane, respectively (Christian, Haynes, Phillips, & Rothblat, 1997; Gimpl, Burger, & Fahrenholz, 1997; Zidovetzki & Levitan, 2007).

FIGURE 1.

FIGURE 1

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Two mechanisms of regulation of membrane proteins by cholesterol. Direct cholesterol–protein interactions and indirect regulation trough changes in the bilayer's physical properties. CARC, inverted CRAC sequence; Ch, cholesterol; CRAC, cholesterol recognition amino acid consensus; PL, phospholipids; TMP, transmembrane protein

In contrast to the generalized idea that the activity of membrane proteins that are inhibited by membrane cholesterol depletion, is necessarily stimulated by cholesterol loading or vice versa, we have recently reported that https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=561/https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=562 channels activity was inhibited by both increase and decrease of the cholesterol level in the plasma membrane (Delgado‐Ramirez et al., 2018). These results indicate that an optimum level of cholesterol guarantees the proper functioning of these channels (Figure 2). At present, it is not clear whether or not this is a generalized mechanism exhibited for all transmembrane proteins. Furthermore, it is important to note that such a mechanism could depend on diverse and particular conditions for each membrane protein as type, isoform and localization into or out of membrane rafts. In this review, we refer and discuss a series of studies assessing this possibility.

FIGURE 2.

FIGURE 2

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Critical cholesterol concentration is necessary for the proper activity of plasma membrane proteins. Decrease and enrichment of cholesterol from the basal level result in alteration of membrane protein activity. Here are represented various possibilities of activity modification and representative examples of each one. In the centre of the illustration, the examples mainly discussed along this review are highlighted. Solid lines indicate experimental reported effects and the dashed lines the not yet described putative possibilities. A2A, adenosine https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=19; CCK1, cholecystokinin type 1 receptor; CCK2, cholecystokinin type 2 receptor; GABAA, GABA‐A receptor; Kir, inwardly rectifying K+ channel; KV, voltage‐dependent K+ channel; NCX, Na+/Ca2+ exchanger; Rhodo, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2963; https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=507 , transient receptor potential vanilloid 1

2. K V 7.2/K V 7.3 CHANNELS ARE INHIBITED BY DECREASE OR LOADING OF PLASMA MEMBRANE CHOLESTEROL

Cholesterol regulates the activity of multiple types of ion channels, including K+ channels (Martens et al., 2000; Romanenko, Rothblat, & Levitan, 2002; Hajdú, Varga, Pieri, Panyi, & Gáspár, 2003; Hibino & Kurachi, 2007; D'Avanzo, Hyrc, Enkvetchakul, Covey, & Nichols, 2011; Bukiya et al., 2015; Rudakova, Wagner, Frank, & Volk, 2015). Thus, http://kir3.1/Kir3.4/https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=437 (GIRK or KACh) cardiac channels are stimulated, while https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=430&familyId=74&familyType=IC channels are inhibited by increased plasma membrane cholesterol and vice versa, that is Kir3.1/Kir3.4 are inhibited and Kir2.X are stimulated by cholesterol depletion (Romanenko et al., 2002; Deng et al., 2012). The voltage‐activated https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=540, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=542 and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=546 channels, which are associated with cholesterol‐rich membrane domains (membrane rafts), are also modulated by cholesterol content. Depletion of plasma membrane cholesterol modifies their voltage‐dependent steady‐state activation and inactivation curves (Martens et al., 2000; Martens, Sakamoto, Sullivan, Grobaski, & Tamkum, 2001; Pottosin, Valencia‐Cruz, Bonales‐Alatorre, Shabala, & Dobrovinskaya, 2007).

KV7.2/KV7.3 channels are preferentially expressed in neurons, where they generate the M‐current that stabilizes the membrane potential and controls neuronal excitability (Wang et al., 1998). Recently, we reported that either an increase or decrease of cholesterol in the plasma membrane inhibits the activity of KV7.2/KV7.3 channels expressed in HEK‐293 cells (Delgado‐Ramírez et al., 2018; Figure 2). Interestingly, cholesterol depletion modified the kinetics and voltage‐dependence of KV7.2/KV7.3 channels, whereas cholesterol enrichment did not. Furthermore, our data obtained from three distinct experimental approaches to manipulate the free/bound ratio of membrane cholesterol (depletion by methyl‐β‐cyclodextrin, complex by Filipin III and oxidation of membrane cholesterol by cholesterol oxidase) suggest that under basal conditions, cholesterol stimulates KV7.2/KV7.3 channels via a direct interaction. Related to the inhibitory effect on KV7.2/KV7.3 channels by enrichment of membrane cholesterol level, we propose that higher level cholesterol changes the physical properties of the plasma membrane altering the activity of the channels. To our knowledge, this is the first report establishing that a voltage‐dependent ion channel requires an optimum level of cholesterol in the plasma membrane to maintain its proper functioning. Now, we are exploring the molecular bases of these modulatory effects, by evaluating the putative sites on KV7.2/KV7.3 molecule involved in the direct interaction cholesterol channel.

3. GABAA RECEPTORS POTENCY IS REDUCED BY DECREASE OR ENRICHMENT OF PLASMA MEMBRANE CHOLESTEROL

https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=72 are target for the major inhibitory neurotransmitter in the CNS and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1067 (MacDonald & Olsen, 1994; Chebib & Johnston, 1999). These ionotropic receptors conduct Cl− into the cell upon activation, promoting plasma membrane hyperpolarization (MacDonald & Olsen, 1994; Chebib & Johnston, 1999). Drugs acting on GABAA are widely used in medical practice. These receptors contain distinct modulatory sites for different anxiolytic, hypnotic and anti‐convulsants, such as benzodiazepines and barbiturates and the convulsant https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4051 (Olsen, 2018). The relevance of cholesterol modulation on GABAA was first evaluated on the action of their potentiators such as neurosteroids (Bennett & Simmonds, 1996; Sooksawate & Simmonds, 1998). In rat synaptosomal membranes, cholesterol enrichment reduced the enhancing effect of pregnanolone on https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4193 binding to GABAA (Bennett & Simmonds, 1996). Interestingly, in dissociated hippocampal neurons, increased membrane cholesterol reduced the potentiation of GABA currents by pregnanolone, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4108 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5461 but not by the anaesthetic https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5464 (Sooksawate & Simmonds, 1998). In the same type of neurons, this group (Sooksawate & Simmonds, 2001b) also reported that cholesterol depletion as well as cholesterol enrichment reduced the potency of GABA to activate GABAA receptor currents, causing a shift to the right in the corresponding concentration–response relationships. Remarkably, the shifting was related to the degree of cholesterol depletion or enrichment. Thus, they obtained a bell‐shaped relationship between GABA EC50 and membrane cholesterol level (Figure 2). Furthermore, in an effort to elucidate the mechanism(s) through which cholesterol modulates GABAA receptors, they employed a chiral analogue of cholesterol (epicholesterol). Plasma membrane enrichment with epicholesterol fully mimic the effects of cholesterol loading, that is, reduced the potency of GABA. However, epicholesterol failed to restore GABAA receptor function in previously cholesterol‐depleted cells as cholesterol did, pointing to a specific molecular requirement for cholesterol. The authors propose two different mechanisms to explain the enrichment‐ and depletion‐induced increases in GABA EC50. In basal cholesterol level condition, a direct interaction of cholesterol–GABAA receptors supports agonist‐induced opening. Therefore, the reduction on membrane cholesterol decreases GABA potency. On the other hand, cholesterol (or epicholesterol) enrichment decreases the GABA potency by altering the membrane's physical properties (Figure 1). Recent reports support these conclusions. Binding sites for cholesterol on GABAA receptors have been predicted using docking and molecular dynamic simulations. These models suggest a flexible binding mode for cholesterol on intersubunits sites (Hénin, Salari, Murlidaran, & Brannigan, 2014) and changes in the lipid bilayer elasticity have been also reported to regulate GABAA receptors (Søgaard et al., 2006).

4. GPCR https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=76 SIGNALLING IS BIPHASIC‐DEPENDENT OF PLASMA MEMBRANE CHOLESTEROL

GPCRs are seven transmembrane domains proteins. They are involved in a myriad of diverse cell functions (Simon, Strathmann, & Gautam, 1991; Lefkowitz, 2007). GPCR bind hormones, peptides and neurotransmitters. They activate intracellular signalling through their association with heterotrimeric G proteins. Signalling coupled to GPCR modulates the activity of varied cytosolic and membrane proteins (Simon et al., 1991; Lefkowitz, 2007). Expression, localization and function of GPCR are influenced by plasma membrane composition. For example, both plasma membrane trafficking (Kitson, Mullen, Cogdell, Bill, & Fraser, 2011; Kumar & Chattopadhyay, 2020) and functioning of the GPCR depend on the membrane cholesterol content (Chini & Parenti, 2004; Licon et al., 2015; Gimpl, 2016; Desai & Miller, 2018). This regulatory action on proteins is associated with either cholesterol effects on the biophysical properties of the plasma membrane or direct interaction of GPCR–cholesterol (Cherezov et al., 2007; Wu et al., 2014; Rouviere, Arnarez, Yang, & Lyman, 2017; Desai & Miller, 2018). The cholecystokinin‐1 (CCK1) receptor belongs to Class A GPCR and regulates diverse physiological processes as gallbladder contraction, pancreatic secretion, gastroenteric motility and appetite (Gibbs, Young, & Smith, 1973; Chandra & Liddle, 2007). Cholecystokinin is a hormone synthesized and released by cells of the small intestine (Gibbs et al., 1973; Chandra & Liddle, 2007). CCK1 receptor couples predominantly to Gq/11 protein signalling that includes https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=274‐β activation and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4222 generation, the release of DAG and intracellular calcium, and the activation of https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=286&familyType=ENZYME. Gq/11 protein signalling regulates the activity of numerous cytosolic and transmembrane proteins. CCK1 receptors, similar to KV7.2/KV7.3 channels and GABAA receptors, previously referred to, exhibit a particular sensitivity to plasma membrane cholesterol content, where both depletion and increase of cholesterol inhibit their activity (Desai et al., 2016; Desai & Miller, 2018; Potter, Harikumar, Wu, & Miller, 2012; Harikumar et al., 2005). Harikumar et al. (2005) and Potter et al. (2012) showed that cholesterol depletion significantly decreased the agonist binding affinity to CCK1 receptors, while enrichment of plasma membrane cholesterol content actually increased CCK1 binding affinity. However, this increased binding evoked a decreased signalling, as observed when the membranes are cholesterol depleted (Figure 2). Importantly, the defective response of CCK1 receptors induced by cholesterol depletion was corrected upon cholesterol repletion. The authors concluded that a defective coupling of the CCK1 receptor to its Gq/11 protein signalling results from the altered plasma membrane cholesterol content. Interestingly, the closely related https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=77 was not sensitive to cholesterol (Potter et al., 2012; Desai, Harikumar, & Miller, 2014). This functional difference was used to generate chimeric CCK1/CCK2 receptor constructs and then to explore the molecular basis for this difference. This approach demonstrates that the effect of cholesterol depletion is mediated by direct interaction of CCK1 receptor–cholesterol with a recognition motif on the transmembrane segment 3 (Potter et al., 2012; Desai et al., 2014). Noteworthy, all these data were obtained in vitro by modifying the membrane cholesterol content of CHO cell lines, engineered to express the wild type human CCK1 or CCK2 receptor and the chimeric CCK1/CCK2 receptor constructs (Desai et al., 2014; Desai et al., 2016; Desai & Miller, 2018; Potter et al., 2012; Harikumar et al., 2005).

5. NA+/CA2+ EXCHANGER ACTIVITY REQUIRES AN OPTIMAL CHOLESTEROL PLASMA MEMBRANE CONCENTRATION

https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=180), another transmembrane protein, transports three Na+ in exchange for one Ca2+, with transport direction dependent on electrochemical gradient of both ions. NCX plays a relevant role in the dynamics of intracellular Ca2+ and cell excitability (Nachshen, Sánchez‐Armáss, & Weinstein, 1986; Sánchez‐Armáss & Blaustein, 1987; Blaustein & Lederer, 1999). In its forward mode of operation, NCX mediates Ca2+ efflux and Na+ influx. However, due to certain events such as membrane depolarization and/or intracellular Na+ accumulation, NCX is able to mediate Ca2+ influx and Na+ efflux (inverse operating mode). Vemuri and Philipson (1988, 1989) studying the activity of the NCX reconstituted into lipid vesicles from cardiac plasma membranes and observed a strong dependence on membrane cholesterol. NCX activity was minimal in the absence of cholesterol and increased to a maximum near the reported physiological cholesterol concentration (Philipson, Bers, & Nishimoto, 1980), beyond which activity decreased again. The corresponding concentration–activity relationship shows a typical bell shape with the peak activity of NCX close to the basal plasma membrane concentration (Vemuri & Philipson, 1988; Vemuri & Philipson, 1989; Figure 2). The authors concluded that the optimum plasma membrane environment for NCX activity is quite sensitive to cholesterol content, but they did not suggest any specific mechanisms to explain such phenomenon.

6. GLUCAGON‐STIMULATED ADENY CYCLASE (AC) ACTIVITY IS VERY SENSITIVE TO CHANGES IN MEMBRANE CHOLESTEROL

https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=257#1280 (ACs) are widespread signalling proteins with key regulatory roles in essentially all cells. They catalyse the conversion of https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1713) to https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2352 and pyrophosphate (Tang & Gilman, 1992). The cAMP produced by AC is an important second messenger, used for intracellular signal transduction, such as transferring into cells the effects of hormones, for example https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1136 (Tang & Gilman, 1992). Glucagon is a peptide hormone released from alpha cells of the pancreas (Müller, Finan, Clemmensen, DiMarchi, & Tschöp, 2017). Glucagon binds and activates the glucagon receptor, a member of the Class B family of GPCRs, coupled to Gs protein signalling that includes AC activation and cAMP production. cAMP triggers the release of glucose from the liver during fasting and thus has an important role in glucose homeostasis (Müller et al., 2017). In two separate papers, Whetton, Gordon, and Houslay (1983a) and Whetton, Gordon, and Houslay (1983b) reported the effect of cholesterol on the glucagon‐stimulated AC from rat liver. They found that elevated or reduced levels of membrane cholesterol both inhibited the AC activity (Figure 2). In both cases, the glucagon‐stimulated activity was affected more than basal AC activity. For elevated cholesterol, the authors suggested that inhibition of the AC activity is due, at least in part, to a decrease in bilayer fluidity mediated by cholesterol. For low cholesterol, the authors speculated that an inhibitory phospholipid released from cholesterol‐rich domains (after cholesterol depletion) can interact and inhibit the enzyme. Interestingly, the https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=940‐stimulated AC activity, from bovine caudate synaptosomal membranes, was inhibited by increased cholesterol levels. However, decreased levels of cholesterol had little effect on the activity of the enzyme (Maguire & Druse, 1989). Taken together, these data suggest that there are differences in the regulation of AC by the cholesterol content, depending on the tissue of origin of the enzyme, the type of receptor coupled to AC, and maybe the location of the enzyme on the membrane, that is, raft versus non‐rafts membrane domains. In fact, recent studies show that five ACs (AC1, AC3, AC5, AC6 and AC8) are associated with membrane caveolar/raft microdomains. On the other hand, four ACs (AC2, AC4, AC7 and AC9) are found within non‐raft plasma domains (Johnstone, Agarwal, Harvey, & Ostrom, 2018).

7. NA+,K+ATPASE

https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=158#834 is a protein complex located on the plasma membrane of animal cells (Lambropoulos et al., 2016). It is a member of the https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=138&familyType=TRANSPORTER class that utilizes ATP to transport three Na+ out of and two K+ into the cell (Thomas, 1972). Na+,K+‐ATPase functioning is implicated in cellular electrochemical gradient maintenance, osmotic balance, excitability in neurons and muscles fibres, cell adhesion and motility (Matchkov & Krivoi, 2016; Cui & Xie, 2017). This ion exchanger exemplifies another membrane protein critically sensitive to plasma membrane cholesterol content (Figure 2). Yeagle (1983) and Yeagle, Young, and Rice (1988) altered the cholesterol content of bovine kidney medulla basolateral membrane at different levels and measured the activity of Na+,K+‐ATPase and found that maximal activity was observed at the basal plasma membrane cholesterol content. In contrast, the activity of this enzyme was inhibited when the membrane cholesterol was either increased above or decreased below that basal content. As a first proposal, they suggested that effect of high cholesterol levels could be due to a specific interaction between cholesterol and the protein (Yeagle, 1983), but in a later report (Yeagle et al., 1988) they alleged instead that there was not enough strong evidence to support this hypothesis. Similar results have been reported by at least three other groups (Kutryk & Pierce, 1988; Sotomayor et al., 2000; Garcia et al., 2019). The first authors, evaluated the activity of Na+,K+‐ATPase in cardiac sarcolemmal from dog ventricles when variations in membrane cholesterol content were tested and observed a small but significant reduction in the activity of the enzyme, 27% or 18% related to control value when the basal membrane cholesterol level was decreased or increased, respectively. No major explanation for this effect was proposed by the authors. Sotomayor et al. (2000) observed also this biphasic modulatory effect of cholesterol in pig kidney basolateral membrane Na+,K+‐ATPase and suggested that changes in the biophysical properties and water content of the plasma membrane were responsible for the effect. In particular, these authors suggest that hydration at the protein–lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. The presence of water at the protein–lipid interface stressed the possible role of hydration as a factor influencing membrane protein structure and in turn its activity (Sotomayor et al., 2000). Importantly, it is also known that the water permeability decreases with relative cholesterol concentration in thin lipids membranes (Finkelstein & Cass, 1967). More recently, Garcia et al. (2019) working on membrane fragments from pig kidney outer medulla similarly concluded that an optimal cholesterol level within the membrane is required to maximize Na+,K+‐ATPase activity. Interestingly, the authors performed also molecular dynamic simulations which predicted multiple cholesterol‐binding sites on Na+,K+‐ATPase. However, they suggest that cholesterol primary influence was on the mechanical properties of plasma membrane via its modulation of local bilayer shape, rather than relative stabilization through preferential binding.

8. FINAL CONSIDERATIONS

Cholesterol is the preeminent lipid in the plasma membrane of animal cells. Then, controlling cholesterol membrane level is critical to support both the basal membrane biophysical properties and the normal activity of several integral transmembrane proteins. Changes in such cholesterol homeostatic level could evoke altered activity of key membrane proteins, which probably need a critical cholesterol level in their surrounding plasma membrane for their proper functioning. A potential physiological benefit of such critical cholesterol level is related to the presence of a cholesterol gradient along the trafficking/targeting pathway of diverse plasma membrane proteins. In the endoplasmic reticulum, the cholesterol content is low (typically 3–6 mol% of lipids) and gradually increases through the Golgi complex to finally reach a high level at the plasma membrane (30–40 mol% of lipids; Ikonen, 2018). Thus, a specific cholesterol content requirement for the optimal activity of a given membrane protein could keep it at very low (if any) functional level, acting as a safety mechanism to control the protein activity in sites where it is not necessary or required. A similar regulatory mechanism has been proposed to explain the control of the activity of ion transporters and channels during biosynthesis or vesicle trafficking by https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2387. Low PIP2 concentrations in the secretory pathway would inactivate all of the systems that are stimulated by PIP2 in the plasma membrane (Hilgemann, Feng, & Nasuhoglu, 2001; Hilgemann, 2012; Kruse & Hille, 2013).

A common cholesterol‐lowering therapy involves the use of long‐term medications (e.g. statins), in order to avoid cardiovascular disease events (Greenfeder, 2009). However, given the critical level of membrane cholesterol for the proper function of diverse proteins, this kind of pharmacological treatments is expected to interfere in several and varied cellular processes (Wainwright, Mascitelli, & Goldstein, 2009). It is well known, for instance, that pharmacological inhibition of the biosynthesis of cholesterol by statins, in murine and human pancreatic β cells, impairs https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5012 secretions by affecting the functioning of https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=80 and the fusion process of insulin secretory granules (Xia et al., 2008). Likewise, chronic cholesterol depletion by statins modifies the trafficking (Kumar & Chattopadhyay, 2020) and reduces the level of specific ligand binding and G‐protein coupling in https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1 (Shrivastava, Pucadyil, Paila, Ganguly, & Chattopadhyay, 2010). In the same direction, as we have mentioned before, some pharmacological properties of the GABAA receptors are also influenced by the membrane cholesterol level (Sooksawate and Simmonds, 2001a), which could be relevant after a cholesterol‐lowering therapy. More translational investigation is necessary to deeper assess these questions in the future.

9. CONCLUDING REMARKS

  • In animal cells, cholesterol is important for efficient function of the proteins in its native membrane environment.

  • A right amount of cholesterol guarantees the proper functioning of several membrane proteins.

  • Cholesterol modulation on transmembrane proteins exhibits a monotonic or a biphasic dependence of cholesterol content (Figure 2).

  • The biphasic dependence of cholesterol content is present in diverse cell types or membrane models (Table 1).

  • Two mechanisms are proposed to explain the modulatory effect of cholesterol: direct interaction protein–cholesterol and by alteration of biophysical membrane properties (Figure 1).

  • Cholesterol modulation for diverse membrane proteins has not previously been performed at cholesterol contents significantly lower or higher than found in the native membrane during the same study. Therefore, such effect has not been reported or even explored and require further investigation.

  • The established long‐term cholesterol‐lowering therapies (e.g., statins treatment) could interfere in several and varied cellular processes varied cellular processes, affecting the optimal function.

TABLE 1.

Biphasic modulation of membrane proteins by cholesterol

Protein Cell type model Effect of membrane cholesterol Reference
KV7.2/KV7.3 channel HEK293 cells Both depletion (~50%) and increase (ND) of cholesterol decreased the activity Delgado–Ramírez et al. (2018)
GABAA receptor Acutely dissociated rat hippocampal neurons Both depletion (~56%) and increase (~82%) of cholesterol reduce potency of GABA Sooksawate and Simmonds (2001b)
G protein‐coupled cholecystokinin receptor CHO cells Both depletion (~40%) and increase (~40%) of cholesterol decreased peak intracellular Ca2+ response Harikumar et al. (2005)
Na+/Ca2+ exchanger Carrier reconstituted in vesicles from cardiac plasma membranes Both depletion (~75%) and increase (~50%) of cholesterol decreased the activity Vemuri and Philipson (1988) and Vemuri and Philipson (1989)
AC Rat liver plasma membranes Both depletion (~55%) and increase (~31%) of cholesterol decreased the activity Whetton et al. (1983a) and Whetton et al. (1983b)
Na+‐K+ ATPase Cardiac sarcolemmal from dog ventricles/ bovine kidney medulla/basolateral membrane vesicles/membranes from outer medulla of pig kidney Both depletion (~17–56%) and increase (~37–96%) of cholesterol decreased the activity Kutryk and Pierce (1988), Yeagle et al. (1988), and Sotomayor et al. (2000)
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Abbreviation: ND, not determined.

9.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos et al., 2019; Alexander, Fabbro et al., 2019; Alexander, Kelly et al., 2019; Alexander, Mathie et al., 2019).

AUTHOR CONTRIBUTIONS

U.M., S.S.‐A., and A.A.R.‐M. designed the review. M.D.‐R., C.R.‐M., A.A.R.‐M., S.S.‐A., and U.M. wrote the final version of the manuscript. All authors reviewed the manuscript and approved it for publication.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheresto the principles for transparent reporting and scientific rigour ofpreclinical research as stated in the BJP guidelines for Design & Analysis,and Animal Experimentation, and as recommended by funding agencies, publishers,and other organizations engaged with supporting research.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

ACKNOWLEDGEMENTS

This work was partially supported by grants from CONACyT to A.A.R.‐M. (CB‐284443) and to A.A.R.‐M., S.S.‐A., and U.M. (IFC‐2016‐1955) and from Universidad Autónoma de San Luis Potosí to A.A.R.‐M. (C18‐FRC‐08‐03.03) and to U.M. (C19‐FAI‐05‐59.59). We thank Nohelia Meza‐Meza for helpful suggestions on the manuscript.

Meza U, Delgado‐Ramírez M, Romero‐Méndez C, Sánchez‐Armass S, Rodríguez‐Menchaca AA. Functional marriage in plasma membrane: Critical cholesterol level–optimal protein activity. Br J Pharmacol. 2020;177:2456–2465. 10.1111/bph.15027

Contributor Information

Ulises Meza, Email: umeza@uaslp.mx.

Aldo A. Rodríguez‐Menchaca, Email: aldo.rodriguez@uaslp.mx.

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