Cholesterol is a major lipid component of the plasma membrane in mammalian cells constituting up to 45 mol % with respect to other lipids [1, 2]. Yet, even a limited increase in blood and/or tissue cholesterol of up to 2-3 fold above the physiological level is cytotoxic [1-3] and is associated with the development of cardiovascular disease [4-6]. The underlying source for the effect of cholesterol on cellular functions is its ability to alter the function of multiple membrane proteins including ion channels (see, for example, reviews [7-9]).
In recent years, high cholesterol diet has been shown to affect the function of multiple ion channels. In this chapter we focus on the effect of dietary-induced increase in blood and tissue cholesterol levels on potassium channels. Potassium channels are among the largest and most complex types of ion channels. They are widely expressed in human tissues and are involved in many aspects of cell function including membrane excitability, regulation of heart rate, neuronal signaling, vascular tone, insulin release and salt flow across epithelia (see, for example, reviews [10-17]). In addition, they also play a critical role in the protection of neurons and muscle under metabolic stress. As a result, mutations in potassium channels lead to a wide range of disease in the brain (epilepsy, episodic ataxia), ear (deafness), heart (arrhythmia), muscle (myokymia, periodic paralysis), kidney (hypertension), pancreas (hyperinsulinemic hypoglycemia, neonatal diabetes). Therefore, the effect of hypercholesterolemia on potassium channel function has important pathophysiological implications.
The most common effect of a high-cholesterol diet on ion channels in general and potassium channels in particular is a decrease in channel activity. Yet, the activity of some channels is increased following a high cholesterol diet. For example, hypercholesterolemia suppressed the function of the Kir2 subfamily of inwardly rectifying potassium (Kir) channels in different cell types by ~2 fold [18-19]. However, atrial G-protein gated inwardly rectifying potassium channels (GIRK or Kir3) that underlie KACh currents in the heart are enhanced by a high-cholesterol diet . Several types of voltage gated (Kv) channels were sensitive to changes in the level of cellular membrane and dietary cholesterol [20-26]. The majority of the reports described suppression of channel function by high-cholesterol diet. Moreover, large conductance calcium-activated potassium (BK) channels were often suppressed following a high-cholesterol diet [27-31].
In this chapter, we will describe the implications of high-cholesterol dietary intake on members of three major families of potassium channels: voltage gated potassium (Kv) channels, calcium-activated potassium (KCa) channels of large conductance (BK) and inwardly rectifying potassium (Kir) channels. We will demonstrate that not only does high-cholesterol diet increase the levels of blood cholesterol but it also increases the level of cholesterol in tissues in which these types of channels are expressed. We will show that this cholesterol accumulation
2. High-cholesterol diet model
Cholesterol-rich diet that is characteristic of Western societies critically controls blood lipid levels in several species, including humans [32-33]. Regression models have been reported for serum total cholesterol, triacylglycerol, and low-density-, high-density-, and very-low-density-lipoprotein cholesterol. In particular, correlations between increased levels of dietary cholesterol and these plasma lipids and lipoproteins were found to be 0.74, 0.65, 0.41, 0.14, and 0.34, respectively . It has been predicted that compliance with the dietary recommendation to consume <300 mg cholesterol per day (with 30% of energy from fat, < 10% from saturated fat) will reduce plasma total and low-density-lipoprotein-cholesterol (LDL) concentrations by approximately 5% compared with amounts associated with the average American diet . Restriction of dietary cholesterol intake represents a widely used preventative measure against numerous pathological conditions since increased total cholesterol and LDL levels are well-recognized risk factors for several largely prevalent pathologies, including stroke [34-37], coronary heart disease [38-39], vascular dementia , and atherosclerosis . Therefore, it is not surprising that a cholesterol-rich diet has been recreated in a research laboratory setting to study the deleterious effects of cholesterol-rich food intake.
High-cholesterol diet-induced hypercholesterolemia is widely used for studies on monkeys , hamsters , guinea pigs , rabbits [20, 27, 29, 45, 46], rats [19, 47-49] and mice . The dietary-induced hypercholesterolemia model has several advantages. First, it mimics the high-cholesterol food intake that is characteristic to the US population, and which impacts cholesterol levels in the blood of human individuals [32-33]. Second, it does not require alteration of the genetic background of the animal. These advantages make high-cholesterol diet a useful tool to manipulate cholesterol levels in species in which genetic alterations to achieve hypercholesterolemia are challenging. For example, diet-induced changes in blood cholesterol level have been detected in primates besides humans, such as baboons
It should be noted that earlier studies have documented the existence of “hyper-“ and “hyporesponders” to a cholesterol-rich diet in the human population. Trials with the same subjects demonstrated that the human population includes people with a consistently low or high response to increased dietary intake of cholesterol (reviewed by ). Similarly to humans, other primates also vary in their blood lipid responses to dietary lipid composition. Selective breeding of primates based on their individual responses to the composition of the diet resulted in lines that are characterized by low versus high responses to changes in dietary lipids. Thus, similarly to humans, changes in lipoprotein patterns in response to dietary cholesterol seem to be heritable in primates (reviewed by ). Further studies have shown that the differential response to cholesterol consumption in smaller laboratory animals also results in inbred strains of rabbits, rats, and mice that differ in their sensitivity to high-cholesterol diet. Their responsiveness to high-cholesterol diet is largely influenced by the genetic background . Compared to humans, changes in blood cholesterol and LDL levels induced by high-cholesterol diet in lab animals are robust and of high magnitude. Therefore, a high-cholesterol diet represents a useful and practical tool to induce an increase in blood cholesterol levels that ultimately leads to hypercholesterolemia in animal models.
In a typical protocol for a high-cholesterol diet in animal models, the animal would be fed (
In our rat model of a high-cholesterol diet, male Sprague-Dawley rats (50 g) were subjected to an
3. Effect of high-cholesterol diet on potassium channels.
In view of the advantages of a high cholesterol diet described above and its ability to adequately represent the characteristics of high cholesterol food intake in the US, it is widely used for studies on ion channel function during dyslipidemia and hypercholesterolemia. For instance, dietary-induced hypercholesterolemia was shown to up-regulate the function of L-type Ca2+-channels in detrusor smooth muscle , transient receptor potential channels 5 and 6 (TRPC5 and TRPC6) in aortic endothelial cells , cardiac G protein gated inwardly rectifying potassium channels , and epithelial Na+ channels (eNaC) .
In this chapter, we will focus on high-cholesterol diet-driven changes in the function of potassium channels. In particular, we will discuss the effect of an increase in the dietary intake of cholesterol on voltage gated (Kv) channels, calcium activated potassium channels (KCa) and inwardly rectifying potassium channels (Kir).
High-cholesterol diet failed to modulate KV channel function after a brief placement of an Ossabaw miniature swine model on a high-fat/high-cholesterol/high-fructose diet . The animals were fed by the diet for 9 weeks. The authors considered the duration of diet administration to be relatively short. As a result, only an early stage of a complex metabolic syndrome developed. The diet caused ~4-fold increase in blood cholesterol level in the group on diet compared to the control group on standard chow. Coronary arterioles from both groups were isolated and pressurized to 60 cmH2O for
A more complex scenario was observed in work by Heaps
These data showing a reduced KV component in the whole-cell outward potassium current are consistent with another report that focused on potassium currents in swine coronary artery smooth muscle cells . The animals were placed on a high-fat diet for 20 weeks. The diet significantly increased the total blood serum cholesterol level and triglycerides. Remarkably, the increase in both blood lipid components was higher in female swines. 4-aminopyridin-sensitive component of the whole-cell outward potassium current recorded from the isolated coronary artery smooth muscle cells was significantly diminished in the high-cholesterol diet group of male swines. In females, however, no significant reduction in KV 4-aminopyridine-sensitive (KV component) was detected . This report suggests that the effect of high-cholesterol diet on the function of KV channels may be gender-specific.
Loss of KV channel current and its contribution to vasodilatory responses has not only been documented in coronary arteries, but also in the
Apart from the vascular system, the effect of a high-cholesterol diet on KV channels was studied in the
Several animal models have been used to document that plasmalemma BK channels are sensitive to dietary-induced hypercholesterolemia . Considering the ample evidence linking hypercholesterolemia to cardiovascular disease, and the key role of BK channels in regulating vascular tone , most of the studies describe the effect of a high-cholesterol diet on BK channel function in the vascular system. One of the early studies addressed changes in endothelium-dependent and independent components
Hypercholesterolemia-driven increase in arterial KCa channel function compared to control chow was also reported in diabetic pigs receiving high-fat/high-cholesterol (2%) diet . Patch-clamp recording of whole-cell outward potassium currents revealed increased density of the KCa-component in the high-cholesterol diet group. Western blot failed to detect a significant increase in the amount of the KCa pore-forming protein. In addition, intracellular calcium concentration did not differ in control versus high-cholesterol diet groups. The data indicated that diabetic hypercholesterolemia leads to an increased functional coupling between KCa and intracellular calcium release.
In contrast to the above
Another study using hypercholesterolemic rabbits to test acetylcholine-induced vasorelaxation focused on renal artery. Rabbits were subjected to a high-cholesterol diet (0.5% cholesterol) for 5 weeks. This diet resulted in an over 50 fold increase in the total blood cholesterol and an almost 40-fold increase in LDL-cholesterol . Contrary to the findings in the hindlimb circulation, acetylcholine-induced dilation of phenylephrine pre-constricted renal arteries was not changed by the high-cholesterol diet. However, the NO-independent (N(G)-nitro-l-arginine-resistant) component of this relaxation was significantly enhanced in arteries from hypercholesterolemic animals. This component totally vanished after endothelial removal in both control and hypercholesterolemic groups, yet was only reduced significantly in the hypercholesterolemic group when an artery with endothelium was incubated in BK and the intermediate conductance KCa channel blocker charybdotoxin .
Studies on rat cerebral arteries yielded results that are in agreement with the conclusions obtained in the hindlimb of rabbits. Specifically, rat middle cerebral arteries obtained from a Sprague-Dawley strain on control versus high-cholesterol (2% cholesterol supplement for either 10 or 18-23 weeks) were dissected, de-endothelialized, cannulated and pressurized at 60 mm Hg. A blood lipid profile revealed a significant increase in the total serum cholesterol, LDL, and triglyceride levels only at 18-23 weeks of diet (Figure 1). The arterial responses to a depolarizing solution containing 60 mM KCl were similar in control and in all hypercholesterolemic arteries. However, treatment of arteries from either one of the high-cholesterol diet groups with the selective BK channel blocker paxilline resulted in vasoconstriction that was significantly smaller compare to the control group (Figure 3A). BK channel function seemed to be altered rather selectively: arterial diameter responses to the KV channel blocker 4-aminopyridine were similar in control versus hypercholesterolemic animals (Figure 3A). First, these results demonstrated that the general contractile capability of the artery (as tested by a high KCl-containing solution) was largely preserved during the high-cholesterol diet. Second, endothelium-independent vasodilation that is mediated by the activity of smooth muscle BK channels was diminished during hypercholesterolemia [30, 31]. Moreover, the fact that reduced sensitivity to paxilline was observed after 10 weeks on a high-cholesterol diet, well before the changes in the blood lipid profile took effect (Figure 1) suggested that BK channels were highly sensitive to dietary cholesterol levels, independent of the increase in blood cholesterol.
Further experimentation took place in an effort to unveil molecular mechanisms that enable the sensitivity of BK channels to dietary cholesterol. First, it was shown that cholesterol accumulation in the wall of de-endothelialized cerebral arteries of hypercholesterolemic rats followed the pattern of increase in blood cholesterol level. In particular, the cholesterol level in cerebral artery tissue was only increased significantly during weeks 18-23 on diet, but not earlier (Figure 3B) . Therefore, the direct accumulation of cholesterol in the vicinity of the BK channel might not be the sole reason for depressed BK channel sensitivity to paxilline during a high-cholesterol intake. The reduction in paxilline-induced cerebral artery constriction by hypercholesterolemia might result from a decreased number of BK channels in arterial smooth muscle. In particular, hypercholesterolemia may down-regulate accessory, smooth muscle-type β subunit (β1) (Figure 2A). Indeed, cerebral arteries of β1 (
Remarkably, dietary cholesterol does not only alter the paxilline-sensitivity of the channel but also protects against ethanol-induced BK channel-mediated constriction of cerebral arteries. It was shown that BK channels represent a major target for ethanol in the cerebral vessels. Upon ethanol application, BK channel function is diminished, and cerebral artery constriction is observed . The protective role of a high-cholesterol diet against alcohol-induced constriction of cerebral arteries was demonstrated
The effect of a high-cholesterol diet on Kir2 channels has been determined in different animal models and cell types. In earlier studies of Kir2 channels expressed in endothelial cells, hypercholesterolemia was induced by administering an atherogenic diet (0.5% cholesterol, 10% lard, and 1.5% sodium cholate) to castrated male Yorkshire pigs . The properties of endothelial Kir2 channels and the values of the membrane potentials were compared in porcine aortic endothelial cells freshly isolated from the pig aortas. Cells isolated from hypercholesterolemic animals had significantly lower Kir currents than those isolated from control cells. Moreover, the membrane potential in hypercholesterolemic pigs was significantly more depolarized compared with that in control animals. More recently, the effect of a high-cholesterol diet on Kir2 channels expressed in cardiac myocytes was determined using a rat model . In these experiments a group of 25-day-old male Sprague-Dawley rats was placed on a high-cholesterol diet (2% cholesterol in standard rodent food). Another group of the same age was fed an isocaloric, cholesterol-free diet from the same supplier. The rats were sacrificed as atrial tissue donors after 20-24 weeks on control or high-cholesterol diet. Notably, as a result of the high-cholesterol diet, in addition to an approximately 2.5-fold increase in serum LDL levels (see Figure 1), there was also an approximately1.8-fold increase in cholesterol levels in the atrial tissue itself (see Figure 5A). This increase in cholesterol levels in the atrial myocytes following the high cholesterol diet resulted in an approximately 60% decrease in Kir2 currents in atrial cardiomyocytes .
In addition to Kir2 channels, atrial myocytes also express Kir3 channels. In particular, atrial KACh channels are heterotetrameric proteins that consist of Kir3.1 and Kir3.4 . Recent studies  demonstrated that unexpectedly rats that were on a high-cholesterol diet for 18-22 weeks exhibited up to 2-3 fold increase in KACh currents that were sensitive to the selective IK, ACh-blocker tertiapin (Figure 5B-5E). The summary data in Figure 5D-E show that the high-cholesterol diet affected both inward and outward currents in a similar manner. Thus, while the effect was more visible for the larger inward currents, the physiologically relevant smaller outward currents were also significantly affected by cholesterol. This result was surprising because an increase in channel function following an increase in cholesterol levels (as shown in Figure 5A) is rare. These data suggest that an increase in cholesterol levels in atrial myocytes may underlie the increase in KACh currents in hypercholesterolemic rats.
Several studies were also carried out to determine the effect of high-cholesterol diet on Kir6 channels. ATP-sensitive K+ (KATP) channels are expressed in the sarcolemma of cardiomyocytes  and in the mitochondrial inner membrane . Structurally, KATP channels are comprised of a pore forming Kir channel (Kir6.1 or Kir6.2) and an ATP-binding regulatory subunit, the sulfonylurea receptor (SUR1, SUR2A, or SUR2B).
Activation of KATP channels mediates coronary vasodilation during decreases in perfusion pressure within the autoregulatory range  and dilation of collateral and noncollateral vessels during ischemia . However, dilation in response to the KATP channel activator aprikalim was not altered in monkeys following an atherogenic diet and reduction in dietary cholesterol .
In contrast, acidosis-induced coronary arteriolar dilation was impaired in hypercholesterolemic rabbits. When myocardial ischemia takes place , the interstitial pH of the heart rapidly decreases followed by an immediate decrease in coronary resistance by microvascular dilation . It was shown that acidosis-induced coronary arteriolar dilation is mediated via the activation of pertussis toxin-sensitive G protein and consequent opening of the KATP channel [81-82]. Since hypercholesterolemia produces structural and functional abnormalities in blood vessels , its impact on coronary microvascular response to acidosis was investigated . Coronary arterioles isolated from rabbit hearts were cannulated to micropipettes in a vessel chamber and microvascular responses were observed. The effect of the KATP channel blocker glibenclamide on the acidosis-induced microvascular responses was examined. Coronary arterioles significantly dilated as the pH was reduced and the dilation was significantly inhibited by glibenclamide. In another set of experiments, rabbits were randomly assigned to normal chow or high-cholesterol diet. After 8 weeks, the responses of isolated coronary arterioles to acidosis were examined in the two groups. Acidosis-induced dilation in the high-cholesterol group was significantly attenuated compared to the control group. These data suggest that KATP channels play an important role in the acidosis-induced dilation of rabbit coronary arterioles and that dilation of coronary arterioles is impaired in hypercholesterolemia. Notably, the impairment occurs upstream of KATP channel opening.
KATP channels play a key role in endogenous cardioprotective mechanisms [85-88]. Specifically, during cardiac ischemia, the levels of intracellular ATP may decrease. This would result in the opening of KATP channels that operate as molecular biosensors for coupling cellular energy metabolism and excitability . The opening of KATP channels leads to increased influx of K+, which then leads to shortening of the action potential duration and to reduction of the Ca2+ overload that occurs during ischemia-reperfusion induced injury [90-92]. Since hyperlipidemia has been shown to interfere with cardioprotective mechanisms, studies were carried out to investigate the interaction of hyperlipidemia with cardioprotection induced by pharmacological activators of KATP channels . Hearts isolated from rats fed a 2% cholesterol-enriched or normal diet for 8 weeks were subjected to 30 min of global ischemia and 120 min of reperfusion in the presence or absence of KATP modulators. In normal diet-fed rats, activation of KATP channels either by the nonselective KATP activator cromakalim or the selective mitochondrial KATP channel opener diazoxide significantly decreased infarct size compared with vehicle-treated control rats. Moreover, the cardioprotective effect was abolished by blocking the channels using the nonselective KATP blocker glibenclamide or the selective mitochondrial KATP channel blocker 5-hydroxydecanoate. In contrast, in cholesterol-fed rats, the cardioprotective effect was not observed following administration of KATP channel activators, demonstrating that cardioprotection by KATP channel activators is impaired in cholesterol-enriched diet-induced hyperlipidemia. Notably, whereas protein levels of Kir6.1 and Kir6.2 remained unchanged, cardiac expression of Kir6.1 was significantly downregulated in cholesterol-fed rats.
Together, these data demonstrate a wide range of effects of a high-cholesterol diet on the function of inwardly rectifying potassium channels and on their physiological implications. Whereas the function of Kir2 and Kir6 channels was suppressed in several cases following a high-cholesterol diet, atrial Kir3 channels were enhanced. Moreover, in the case of Kir6 channels, whereas KATP-mediated coronary vasodilation was not altered in atherosclerotic monkeys, a high-cholesterol diet resulted in impaired cardioprotection by KATP channel activators in rats and impaired KATP-mediated acidosis-induced coronary arteriolar dilation in rabbits.
4. Conclusive remarks
Different types of potassium channels have been shown to be affected by high-cholesterol diet in a variety of species. The modulation of potassium channel activity by high-cholesterol diet results in alterations of organ function
This work was supported by a Scientist Development Grant 11SDG5190025 from the American Heart Association (to A.R.-D.), the Alcoholic Beverage Medical Research Foundation grant for New Investigator (A.B.), and NIH Support Opportunity for Addiction Research for New Investigators R03 AA020184 (A.B.)