**1.6 Cholesterol and ion channels**

A number of studies has demonstrated that the level of membrane cholesterol regulate the ion channel functions *(Levitan et al., 2010; Maguy et al., 2006; Martens et al., 2004). T*he impact of cholesterol on different types of ion channels is highly heterogeneous. In most of the cases cholesterol supresses channel activity that may include decrease in the open probability, unitary conductance and/or the number of active channels on the membrane. This effect was observed in several types of K+ channels, voltage-gated Na+ and Ca+2 channels, as well as in volume-regulated anion channels. However, there are also several types of ion channels, such as epithelial Na+ channels (eNaC) and transient receptor potential (Trp) channels that are inhibited by the removal of membrane cholesterol. Finally, in some cases changes in membrane cholesterol affect biophysical properties of the channel such as the voltage dependence of channel activation or inactivation. Clearly, therefore, more than one mechanism has to be involved in cholesterol-induced regulation of different ion channels.

Studies from our laboratory have shown that many major and important ion channels are regulated by changes in the level of membrane cholesterol (Epshtein et al., 2009; Levitan, 2009; Levitan et al., 2010; Rosenhouse-Dantsker et al., 2011; Singh et al., 2009b; Singh et al., 2011). In general three different mechanisms may be involved in regulation of ion channels by cholesterol: (i) specific interactions (ii) changes in the physical properties of the membrane bilayer and (iii) maintaining the scaffolds for protein-protein interactions. Furthermore, our recent data for the first time demonstrate a specific cholesterol-channel binding (Singh et al., 2011). One possibility is that cholesterol may interact directly and specifically with the transmembrane domains of the channels protein. Direct interaction between channels and cholesterol as a boundary lipid was first proposed in a "lipid belt" model by (Marsh and Barrantes, 1978) suggesting that cholesterol may be a part of a lipid belt or a "shell" constituting the immediate perimeter of the channel protein. Moreover, studies from our laboratory demonstrated that inwardly-rectifying K+ channel are sensitive to the chiral nature of the sterol analogue providing further support for the hypothesis that sensitivity of these channels to cholesterol can be due to specific sterol-protein interactions (Romanenko et al., 2002). An alternative mechanism proposed by Lundbaek and colleagues (Lundbaek and Andersen, 1999) suggested that cholesterol may regulate ion channels by hydrophobic mismatch between the transmembrane domains and the lipid bilayer. More specifically, it was proposed that when a channel goes through a change in conformation state within the viscous medium of the lipid membrane it may induce deformation of the lipid bilayer surrounding the channel. If this is the case, then a stiffer less deformable membrane will increase the energy that is required for the transition (Levitan et al., 2010). It is important to note that these mechanisms are not mutually exclusive. A lipid shell surrounding a channel may also affect the hydrophobic interactions between the channels and the lipids and increase the deformation energy required for the transitions between closed and open states. Finally, obviously, cholesterol may also affect the channels indirectly through interactions with different signalling cascades.

#### **1.7 Cholesterol and oxysterol as major oxidized component in oxidized LDL**

Oxidation of LDL is considered as the major risk factors for the development of coronary artery disease (CAD) and plaque formation (reviewed in (Berliner et al., 2001; Levitan and Shentu, 2011). Indeed, elevated levels of oxLDL are associated with an increased risk of CAD (Toshima et al., 2000) and correlate with plasma hypercholesterolemia both in humans (van Tits et al., 2006) and in the animal models of atherosclerosis (Hodis et al., 1994; Holvoet et al., 1998). It is also well-known that exposure to oxLDL induces an array of proinflammatory and proatherogenic effects but the mechanisms that underlie oxLDL-induced effects remain controversial. Most of studies involving oxLDL are based on ex-vivo oxidation of LDL. The term oxidized LDL is used to describe LDL preparations which have been oxidatively modified *ex vivo* under defined conditions, or isolated from biological sources.

The most typical procedure of LDL oxidation *ex vivo* is incubation of LDL with metal ions, Cu2+ in particular, that leads to the generation of multiple oxidized products in the LDL particle, including oxysterols, oxidized phospholipids, and modified apolipoprotein B (reviewed in (Burkitt, 2001; Levitan and Shentu, 2011). The oxidized LDL preparations described in the literature are broadly divided into two main categories: "minimally modified LDL" (MM-LDL) and (fully or extensively) oxidized LDL (oxLDL) based on the

Studies from our laboratory have shown that many major and important ion channels are regulated by changes in the level of membrane cholesterol (Epshtein et al., 2009; Levitan, 2009; Levitan et al., 2010; Rosenhouse-Dantsker et al., 2011; Singh et al., 2009b; Singh et al., 2011). In general three different mechanisms may be involved in regulation of ion channels by cholesterol: (i) specific interactions (ii) changes in the physical properties of the membrane bilayer and (iii) maintaining the scaffolds for protein-protein interactions. Furthermore, our recent data for the first time demonstrate a specific cholesterol-channel binding (Singh et al., 2011). One possibility is that cholesterol may interact directly and specifically with the transmembrane domains of the channels protein. Direct interaction between channels and cholesterol as a boundary lipid was first proposed in a "lipid belt" model by (Marsh and Barrantes, 1978) suggesting that cholesterol may be a part of a lipid belt or a "shell" constituting the immediate perimeter of the channel protein. Moreover, studies from our laboratory demonstrated that inwardly-rectifying K+ channel are sensitive to the chiral nature of the sterol analogue providing further support for the hypothesis that sensitivity of these channels to cholesterol can be due to specific sterol-protein interactions (Romanenko et al., 2002). An alternative mechanism proposed by Lundbaek and colleagues (Lundbaek and Andersen, 1999) suggested that cholesterol may regulate ion channels by hydrophobic mismatch between the transmembrane domains and the lipid bilayer. More specifically, it was proposed that when a channel goes through a change in conformation state within the viscous medium of the lipid membrane it may induce deformation of the lipid bilayer surrounding the channel. If this is the case, then a stiffer less deformable membrane will increase the energy that is required for the transition (Levitan et al., 2010). It is important to note that these mechanisms are not mutually exclusive. A lipid shell surrounding a channel may also affect the hydrophobic interactions between the channels and the lipids and increase the deformation energy required for the transitions between closed and open states. Finally, obviously, cholesterol may also affect the channels indirectly

through interactions with different signalling cascades.

**1.7 Cholesterol and oxysterol as major oxidized component in oxidized LDL** 

modified *ex vivo* under defined conditions, or isolated from biological sources.

Oxidation of LDL is considered as the major risk factors for the development of coronary artery disease (CAD) and plaque formation (reviewed in (Berliner et al., 2001; Levitan and Shentu, 2011). Indeed, elevated levels of oxLDL are associated with an increased risk of CAD (Toshima et al., 2000) and correlate with plasma hypercholesterolemia both in humans (van Tits et al., 2006) and in the animal models of atherosclerosis (Hodis et al., 1994; Holvoet et al., 1998). It is also well-known that exposure to oxLDL induces an array of proinflammatory and proatherogenic effects but the mechanisms that underlie oxLDL-induced effects remain controversial. Most of studies involving oxLDL are based on ex-vivo oxidation of LDL. The term oxidized LDL is used to describe LDL preparations which have been oxidatively

The most typical procedure of LDL oxidation *ex vivo* is incubation of LDL with metal ions, Cu2+ in particular, that leads to the generation of multiple oxidized products in the LDL particle, including oxysterols, oxidized phospholipids, and modified apolipoprotein B (reviewed in (Burkitt, 2001; Levitan and Shentu, 2011). The oxidized LDL preparations described in the literature are broadly divided into two main categories: "minimally modified LDL" (MM-LDL) and (fully or extensively) oxidized LDL (oxLDL) based on the degree of LDL oxidation. Cu2+ oxidation of LDL can generate both minimally modified and fully oxidized LDLs depending on the duration of the exposure and ion concentration. Two other procedures that are also used to generate oxLDL *ex vivo* are enzymatic oxidation by 15-lypoxygenase or myeloperoxidase or by incubating LDL with 15-lypoxygenase expressing cells (Boullier et al., 2006). It is important to note that while it is controversial whether Cu2+ oxidation occurs *in vivo*, it was shown that there are significant similarities between Cu2+ oxidized LDL and oxLDL found in atherosclerotic lesions (Yla-Herttuala et al., 1989). **However, oxidation of LDL is a complex process that yields an array of bioactive compounds with different biological properties and its composition depends on the degree of LDL oxidation** It is known that oxLDL includes various oxidized products that are divided into neutral lipids, a group that includes cholesterol and cholesterol ester, and phospholipids that include SM, PE, LPC and PC (Subbanagounder et al., 2000). Our recent studies have identififed five major oxysterols in strongly oxidized LDL (16 hours of oxidation data submitted for publication) by the GC analysis: 7α-hydroxycholesterol , 7βhydroxycholesterol, cholesterol 5α,6α- and 5β,6β-epoxides, and 7-ketocholesterol similar to earlier studies (Brown et al., 1996). Our recent unpublished data showed that the most abundant oxysterol is 7β-hydroxycholesterol, followed by 7-ketocholesterol with 7αhydroxycholesterol and cholesterol 5β,6β-epoxide representing minor oxysterols in oxLDL. Two oxysterols, 25-hydroxycholesterol and 27-hydroxycholesterol do not constitute significant components of oxLDL complex, but were found in human atherosclerotic lesions (Bjorkhem et al., 1994; Zidovetzki and Levitan, 2007). 27 hydroxycholesterols, specially is abundant in human atherosclerotic lesions and macrophage-derived foam cells (Brown and Jessup, 1999).

#### **1.8 Impact of oxLDL on cholesterol-rich membrane rafts**

Membrane rafts were originally described as cholesterol- and sphingolipid-rich microdomains that provide platforms for protein-protein interactions in multiple signaling cascades (Brown and London, 1998; Simons and Gerl, 2010; Simons and Ikonen, 1997). Membrane rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes" (Pike, 2006). Most recently, Simons and Gerl (Simons and Gerl, 2010) further defined membrane rafts as "dynamic, nonoscale, sterol-sphingolipid-enriched, ordered assembles of proteins and lipids" that are regulated by specific lipid-lipid, protein-lipid, and protein-protein interactions (Simons and Gerl, 2010). The goal of this section of book chapter is to discuss the recent advances in our understanding of the impact of oxLDL on membrane rafts. Oxysterols are found in abundance in Cu2+-oxidized LDL, in which cholesterol is oxidized preferably at 7 positions resulting in the generation of 7-ketocholesterol, 7*β*hydroxycholesterol, and 7-hydroxycholesterol (Brown and Jessup, 1999). In addition, 27 hydroxycholesterol, that is, generated *in vivo,* has been shown to accumulate in foam cells in atherosclerotic lesions (Brown and Jessup, 1999). Several studies have shown that oxysterols result in inhibition of cholesterol efflux in the mouse and it was suggested that impairment of cholesterol homeostasis by the inhibition of cholesterol efflux may be mechanism by which oxysterols affect cellular function (Kilsdonk et al., 1995; Terasaka et al., 2008). Interestingly, 7-ketocholesterol was shown to deplete cholesterol specifically from the raft domains in human macrophages (Gaus et al., 2004) and disrupt lipid packing of the immunological synapses in sterol-enriched T lymphocytes (Rentero et al., 2008) and in cholesterol-rich membrane domains in endothelial cells (Shentu et al., 2010). These observations suggest that incorporation of oxysterols may also play an important role in oxLDL-induced disruption of cholesterol-rich membrane domains.

#### **1.9 Manipulation of cholesterol in cellular system (***in vitro* **and** *in vivo* **models)**

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes (Zidovetzki and Levitan, 2007). Under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. Here, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.

The high affinity of methyl-β-cyclodextrin (MβCDs) for cholesterol can be used not only to remove cholesterol from the biological membranes but also to generate cholesterol inclusion complexes that donate cholesterol to the membrane and increase membrane cholesterol level. MβCD-cholesterol inclusion complexes are typically generated by mixing cholesterol suspension with a cyclodextrin solution as described earlier (Christian et al., 1997; Klein et al., 1995; Levitan et al., 2000; Zidovetzki and Levitan, 2007). The ratio between the amounts of cholesterol and cyclodextrin in the complex determines whether it will act as cholesterol acceptor or as cholesterol donor (Zidovetzki and Levitan, 2007). The efficiency of cholesterol transfer from MβCD inclusion complex to biological membranes depends on MβCD:cholesterol molar ratio, MβCD-cholesterol concentration, and duration of the exposure (Zidovetzki and Levitan, 2007). Thus, it is important to note that exposing cells to MβCD-cholesterol complexes that contain saturating amounts of cholesterol typically results not just in replenishing cholesterol to control levels but in significant cholesterol enrichment. Cholesterol enrichment is observed even if the cells were first depleted of cholesterol and then exposed to MβCD-cholesterol complexes. ApoE -/- mice cholesterol ester transfer protein (CETP) knock out mice are ideal *in vivo* animal model to study cholesterol biosynthetic pathway and its regulation. Our recent study revealed that WT and ApoE -/ mice fed on normal chow diet for 4 weeks, showing a significant increase in the level of cholesterol in ApoE -/- mice as compared to WT (Shentu et al., 2011).

#### **1.10 Treatment of hypercholesterolemia**

Reductions in circulating cholesterol levels can have profound positive impacts on cardiovascular disease, particularly on atherosclerosis, as well as other metabolic disruptions of the vasculature. Control of dietary intake is one of the easiest and least cost intensive means to achieve reductions in cholesterol. But in most of the alleviated levels of cholesterol cannot be controlled merely with exercise. Drug therapy is very important to

cholesterol-rich membrane domains in endothelial cells (Shentu et al., 2010). These observations suggest that incorporation of oxysterols may also play an important role in

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes (Zidovetzki and Levitan, 2007). Under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. Here, we discuss useful control strategies that may help to verify that the observed effects are due

The high affinity of methyl-β-cyclodextrin (MβCDs) for cholesterol can be used not only to remove cholesterol from the biological membranes but also to generate cholesterol inclusion complexes that donate cholesterol to the membrane and increase membrane cholesterol level. MβCD-cholesterol inclusion complexes are typically generated by mixing cholesterol suspension with a cyclodextrin solution as described earlier (Christian et al., 1997; Klein et al., 1995; Levitan et al., 2000; Zidovetzki and Levitan, 2007). The ratio between the amounts of cholesterol and cyclodextrin in the complex determines whether it will act as cholesterol acceptor or as cholesterol donor (Zidovetzki and Levitan, 2007). The efficiency of cholesterol transfer from MβCD inclusion complex to biological membranes depends on MβCD:cholesterol molar ratio, MβCD-cholesterol concentration, and duration of the exposure (Zidovetzki and Levitan, 2007). Thus, it is important to note that exposing cells to MβCD-cholesterol complexes that contain saturating amounts of cholesterol typically results not just in replenishing cholesterol to control levels but in significant cholesterol enrichment. Cholesterol enrichment is observed even if the cells were first depleted of cholesterol and then exposed to MβCD-cholesterol complexes. ApoE -/- mice cholesterol ester transfer protein (CETP) knock out mice are ideal *in vivo* animal model to study cholesterol biosynthetic pathway and its regulation. Our recent study revealed that WT and ApoE -/ mice fed on normal chow diet for 4 weeks, showing a significant increase in the level of

Reductions in circulating cholesterol levels can have profound positive impacts on cardiovascular disease, particularly on atherosclerosis, as well as other metabolic disruptions of the vasculature. Control of dietary intake is one of the easiest and least cost intensive means to achieve reductions in cholesterol. But in most of the alleviated levels of cholesterol cannot be controlled merely with exercise. Drug therapy is very important to

**1.9 Manipulation of cholesterol in cellular system (***in vitro* **and** *in vivo* **models)** 

oxLDL-induced disruption of cholesterol-rich membrane domains.

specifically to cyclodextrin-induced changes in cellular cholesterol.

cholesterol in ApoE -/- mice as compared to WT (Shentu et al., 2011).

**1.10 Treatment of hypercholesterolemia** 

avoid the cardiovascular effects of high cholesterol levels in such patients. In this section of the book chapter, we have discussed the regulation cholesterol by drugs as well as will review recent work from our laboratory for the regulation of cholesterol by natural products such as tea/green tea, policosanol and garlic compounds.
