This study investigates how changes in the level of cellular cholesterol affect inwardly rectifying K+ channels belonging to a family of strong rectifiers (Kir2). In an earlier study we showed that an increase in cellular cholesterol suppresses endogenous K+ current in vascular endothelial cells, presumably due to effects on underlying Kir2.1 channels. Here we show that, indeed, cholesterol increase strongly suppressed whole-cell Kir2.1 current when the channels were expressed in a null cell line. However, cholesterol level had no effect on the unitary conductance and only little effect on the open probability of the channels. Moreover, no cholesterol effect was observed either on the total level of Kir2.1 protein or on its surface expression. We suggest, therefore, that cholesterol modulates not the total number of Kir2.1 channels in the plasma membrane but rather the transition of the channels between active and silent states. Comparing the effects of cholesterol on members of the Kir2.x family shows that Kir2.1 and Kir2.2 have similar high sensitivity to cholesterol, Kir2.3 is much less sensitive, and Kir2.4 has an intermediate sensitivity. Finally, we show that Kir2.x channels partition virtually exclusively into Triton-insoluble membrane fractions indicating that the channels are targeted into cholesterol-rich lipid rafts.

Activation of an apical

This study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-beta-cyclodextrin or enriched by exposing the cells to methyl-beta-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-beta-cyclodextrin-cholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane.

Membrane potential of aortic endothelial cells under resting conditions is dominated by inward-rectifier K(+) channels belonging to the Kir 2 family. Regulation of endothelial Kir by membrane cholesterol was studied in bovine aortic endothelial cells by altering the sterol composition of the cell membrane. Our results show that enriching the cells with cholesterol decreases the Kir current density, whereas depleting the cells of cholesterol increases the density of the current. The dependence of the Kir current density on the level of cellular cholesterol fits a sigmoid curve with the highest sensitivity of the Kir current at normal physiological levels of cholesterol. To investigate the mechanism of Kir regulation by cholesterol, endogenous cholesterol was substituted by its optical isomer, epicholesterol. Substitution of approximately 50% of cholesterol by epicholesterol results in an early and significant increase in the Kir current density. Furthermore, substitution of cholesterol by epicholesterol has a stronger facilitative effect on the current than cholesterol depletion. Neither single channel properties nor membrane capacitance were significantly affected by the changes in the membrane sterol composition. These results suggest that 1) cholesterol modulates cellular K(+) conductance by changing the number of the active channels and 2) that specific cholesterol-protein interactions are critical for the regulation of endothelial Kir.

A variety of ion channels, including members of all major ion channel families, have been shown to be regulated by changes in the level of membrane cholesterol and partition into cholesterol-rich membrane domains. In general, several types of cholesterol effects have been described. The most common effect is suppression of channel activity by an increase in membrane cholesterol, an effect that was described for several types of inwardly-rectifying K + channels, voltage-gated K + channels, Ca +2 sensitive K + channels, voltage-gated Na + channels, N-type voltage-gated Ca +2 channels and volume-regulated anion channels. In contrast, several types of ion channels, such as epithelial amiloride-sensitive Na + channels and Transient Receptor Potential channels, as well as some of the types of inwardly-rectifying and voltage-gated K + channels were shown to be inhibited by cholesterol depletion. Cholesterol was also shown to alter the kinetic properties and current-voltage dependence of several voltage-gated channels. Finally, maintaining membrane cholesterol level is required for coupling ion channels to signalling cascades. In terms of the mechanisms, three general mechanisms have been proposed: (i) specific interactions between cholesterol and the channel protein, (ii) changes in the physical properties of the membrane bilayer and (iii) maintaining the scaffolds for proteinprotein interactions. The goal of this review is to describe systematically the role of cholesterol in regulation of the major types of ion channels and to discuss these effects in the context of the models proposed. KeywordsIon channels; Cholesterol; Lipid rafts IntroductionDuring the last decade, a growing number of studies have demonstrated that the level of membrane cholesterol is a major regulator of ion channel function (reviewed by Maguy et al., 2006;Martens et al., 2004). It is also becoming increasingly clear that the impact of cholesterol on different types of ion channels is highly heterogeneous. The most common effect is cholesterol-induced decrease in 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.

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP Ͼ Ͼ ATP > ADP Ͼ Ͼ UTP, and removal of external Ca 2؉ dramatically suppressed the initial ATP-induced fluid secretion (ϳ85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X 7 gene were used to evaluate the role of the P2X 7 receptor in nucleotide-evoked fluid secretion. P2X 7 receptor protein and BzATPactivated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X 7 -null mice. Consistent with these observations, the ATP-induced increases in [Ca 2؉ ] i were nearly abolished in P2X 7 ؊/؊ submandibular acinar and duct cells. ATP appeared to also act through the P2X 7 receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X 7 receptor regulates fluid secretion in the mouse submandibular gland.Salivation is a Ca 2ϩ -dependent process (1, 2) primarily associated with the neurotransmitters norepinephrine and acetylcholine, release of which stimulates ␣-adrenergic and muscarinic receptors, respectively. Both types of receptors are coupled to G proteins that activate phospholipase C␤ (PLC␤) during salivary gland stimulation. PLC␤ activation cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and inositol 1,4,5-trisphosphate (InsP 3 ) production. Activation of Ca 2ϩ -selective InsP 3 receptor channels localized to the endoplasmic reticulum of salivary acinar cells increases the intracellular free calcium concentration ([Ca 2ϩ ] i ). 4 Depletion of the endoplasmic reticulum Ca 2ϩ pool triggers extracellular Ca 2ϩ influx and a sustained elevation in [Ca 2ϩ ] i . This increase in [Ca 2ϩ ] i activates Ca 2ϩ -dependent K ϩ and Cl Ϫ channels promoting Cl Ϫ secretion across the apical membrane and a lumen negative, electrochemical gradient that supports Na ϩ efflux into the lumen. The accumulation of NaCl creates an osmotic gradient which drives water movement into the lumen, thus generating isotonic primary saliva. This primary fluid is then modified by the ductal system, which reabsorbs NaCl and secretes KHCO 3 producing a final saliva that is hypotonic (1, 2).Salivation also has a non-cholinergic, non-adrenergic component, the origin of which is unclear (3). In addition to muscarinic and ␣-adrenergic receptors, salivary acinar cells express other receptors that are coupled to an increase in [Ca 2ϩ ] i such as purinergic P2 and substance P receptors. Like muscarinic and ␣-adrenergic receptors, P2 receptor activation leads to a sustained increase in [Ca 2ϩ ] i in salivary acinar cells (4). In contrast, substance P receptor ac...

Depletion of membrane cholesterol and substitution of endogenous cholesterol with its structural analogues was used to analyze the mechanism by which cholesterol regulates volume-regulated anion current (VRAC) in endothelial cells. Depletion of membrane cholesterol enhanced the development of VRAC activated in a swelling-independent way by dialyzing the cells either with GTPγS or with low ionic strength solution. Using MβCD–sterol complexes, 50–80% of endogenous cholesterol was substituted with a specific analogue, as verified by gas-liquid chromatography. The effects of cholesterol depletion were reversed by the substitution of endogenous cholesterol with its chiral analogue, epicholesterol, or with a plant sterol, β-sitosterol, two analogues that mimic the effect of cholesterol on the physical properties of the membrane bilayer. Alternatively, when cholesterol was substituted with coprostanol that has only minimal effect on the membrane physical properties it resulted in VRAC enhancement, similar to cholesterol depletion. In summary, our data show that these channels do not discriminate between the two chiral analogues of cholesterol, as well as between the two cholesterols and β-sitosterol, but discriminate between cholesterol and coprostanol. These observations suggest that endothelial VRAC is regulated by the physical properties of the membrane.

We have recently shown that the IK1 and maxi-K channels in parotid salivary gland acinar cells are encoded by the K Ca 3.1 and K Ca 1.1 genes, respectively, and in vivo stimulated parotid secretion is severely reduced in double-null mice. The current study tested whether submandibular acinar cell function also relies on these channels. We found that the K + currents in submandibular acinar cells have the biophysical and pharmacological footprints of IK1 and maxi-K channels and their molecular identities were confirmed by the loss of these currents in K Ca 3.1-and K Ca 1.1-null mice. Unexpectedly, the pilocarpine-stimulated in vivo fluid secretion from submandibular glands was essentially normal in double-null mice. This result and the possibility of side-effects of pilocarpine on the nervous system, led us to develop an ex vivo fluid secretion assay. Mammals express three major paired salivary glands, the parotid, submandibular and sublingual. The morphology, histology and saliva composition are unique for each gland type (Young & Van Lennep, 1977). These differences are most evident in the secretory endpieces, also known as acini, the cells which secrete the water and most of the protein found in saliva. In rodents, the parotid acinar cells are serous, while submandibular acinar cells are seromucous. In contrast, the secretory endpieces of sublingual glands are mixed in nature, containing both mucous and serous demilune cells. The composition of the saliva collected from individual glands varies according V. G. Romanenko and T. Nakamoto contributed equally to this work. This paper has online supplemental material.to the acinar cell type. Parotid glands produce a watery fluid and sublingual glands secrete a thick, ropey saliva due to the exocytosis of heavily glycosylated mucins, whereas submandibular saliva has an intermediate consistency.The mechanism of fluid secretion is generally studied at two levels: (i) the cellular level gives detailed information under well controlled conditions but is an over-simplified model and (ii) the in vivo level provides access to whole organ function, but the interpretation of results is complicated by systemic effects. In the latter case, saliva secretion is often induced by cholinergic agonists such as pilocarpine. It is well established that pilocarpine can stimulate saliva secretion through glandular muscarinic receptors. However, systemic administration of a cholinergic agonist also activates receptors in the brain and peripheral nervous