Insulin receptor-inspired soluble insulin binder

The insulin receptor (IR) is a 320 kDa membrane receptor tyrosine kinase mediating the pleiotropic actions of insulin, leading to phosphorylation of several intracellular substrates including serine/threonine-protein kinase (AKT1), and IR autophosphorylation. Structural details of the IR have been recently revealed. A high-binding insulin site, L1 (K_d =2 nM), consists of two distant domains in the primary sequence of the IR. Our design simplified the L1 binding site and transformed it into a soluble insulin binder (sIB). The sIB, a 17 kDa protein, binds insulin with 38 nM affinity. The sIB competes with IR for insulin and reduces by more than 50% phosphorylation of AKT1 in HEK 293 T cells, with similar effects on IR autophosphorylation. The sIB represents a new tool for research of insulin binding and signaling properties.     

Actin-dependent clustering of insulin receptors in membrane microdomains

Recent evidence suggests that, after binding insulin, insulin receptors (IR) interact with specialized, cholesterol-containing, membrane microdomains and components of the actin cytoskeleton. Using single particle tracking techniques, we examined how binding of insulin, depletion of membrane cholesterol and disruption of actin filaments affect the lateral diffusion of individual quantum dot-labeled native IR on live rat basophilic leukemia 2H3 cells. We also examined the effects of similar treatments on IR clustering and multivalent insulin binding on these cells using both photon counting histogram analysis and polarization-based fluorescence resonance energy homo-transfer imaging. Our analyses indicate that binding of insulin to IR on these cells is multivalent, involving at least two insulin molecules per IR as labeling concentrations approach 1μM. Insulin binding also reduces lateral diffusion of IR and the size of membrane compartments accessed by IR. For IR that have not bound insulin, lateral diffusion of IR and the size of membrane compartments accessed by IR increase after disrupting actin filaments or depleting membrane cholesterol. However, clustering of insulin-occupied IR is reduced only by disrupting actin filaments or by fixing cells with paraformaldehyde prior to exposure to insulin, but not by depleting membrane cholesterol. Thus, it appears that, although restriction of IR lateral diffusion on these cells is sensitive to both actin filament dynamics and membrane cholesterol content, clustering of insulin-occupied IR primarily involves an actin-dependent mechanism.     

Lack of cholesterol mobilization in islets of hormone-sensitive lipase deficient mice impairs insulin secretion

The observations that hormone-sensitive lipase (HSL) is located in close association to insulin granules in β-cells and that cholesterol ester hydrolase activity is completely blunted in islets of HSL null mice made us hypothesize that the role of HSL in β-cells is to provide cholesterol for the exocytosis of insulin. To test this hypothesis, wild type (wt) and HSL null islets were depleted of plasma membrane cholesterol using methyl-β-cyclodextrin (mβcd). A significant reduction in insulin secretion from HSL null islets was observed whereas wt islets were unaffected. Using synaptosomal protein of 25 kDa (SNAP-25) as indicator of cholesterol-rich microdomains, confocal microscopy was used to show that HSL null β-cells treated with mβcd contained fewer clusters than wt β-cells. These results indicate that HSL plays an important role in insulin secretion by providing free cholesterol for the formation and maintenance of cholesterol-rich patches for docking of SNARE-proteins to the plasma membrane.     

Ligand Binding and G-protein Coupling of the Serotonin1A Receptor in Cholesterol-enriched Hippocampal Membranes

The serotonin_1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Since a large portion of such transmembrane receptors remains in contact with the membrane lipid environment, lipid–protein interactions assume importance in the structure-function analysis of such receptors. We have earlier reported the requirement of cholesterol for serotonin_1A receptor function in native hippocampal membranes by specific depletion of cholesterol using methyl- β-cyclodextrin. In this paper, we monitored the serotonin_1A receptor function in membranes that are enriched in cholesterol using a complex prepared from cholesterol and methyl-β-cyclodextrin. Our results indicate that ligand binding and receptor/G-protein interaction of the serotonin_1A receptor do not exhibit significant difference in native and cholesterol-enriched hippocampal membranes indicating that further enrichment of cholesterol has little functional consequence on the serotonin_1A receptor function. These results therefore provide new information on the effect of cholesterol enrichment on the hippocampal serotonin_1A receptor function.     

An adhesion-based method for plasma membrane isolation: Evaluating cholesterol extraction from cells and their membranes

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-β-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-β-cyclodextrin plasma membrane extraction properties.     

Cholesterol efflux stimulates metalloproteinase-mediated cleavage of occludin and release of extracellular membrane particles containing its C-terminal fragments

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-β-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an ∼80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-β-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-β-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-β-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-β-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and  48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.     

Amyloid toxicity in skeletal myoblasts: Implications for inclusion-body myositis

Skeletal muscle disorder, inclusion-body myositis (IBM) has been known for accumulation of amyloid characteristic proteins in muscle. To understand the biophysical basis of IBM, the interaction of amyloid fibrils with skeletal myoblast cells (SMC) has been studied in vitro. Synthetic insulin fibrils and Aβ_25-35 fibrils were used for this investigation. From the saturation binding analysis, the calculated dissociation constant (K_d) for insulin fibril and Aβ_25-35 fibrils were 69.37 ± 11.17 nM and 115.60 ± 12.17 nM, respectively. The fibrillar insulin comparatively has higher affinity binding to SMC than Aβ fibrils. The competitive binding studies with native insulin showed that the amount of bound insulin fibril was significantly decreased due to displacement of native insulin. However, the presence of native insulin is not altered the binding of β-amyloid fibril. The cytotoxicity of insulin amyloid intermediates was measured. The pre-fibrillar intermediates of insulin showed significant toxicity (35%) as compared to matured fibrils. Myoblast treated with β-amyloid fibrils showed more oxidative damage than the insulin fibril. Cell differentiating action of amyloidic insulin was assayed by creatine kinase activity. The insulin fibril treated cells differentiated more slowly compared to native insulin. However, β-amyloid fibrils do not show cell differentiation property. These findings reinforce the hypothesis that accumulation of amyloid related proteins is significant for the pathological events that could lead to muscle degeneration and weakness in IBM.     

Regulation of heart insulin receptor tyrosine kinase activity by magnesium and spermine

Insulin action and aspects of the insulin-signaling pathway have been studied in the heart although the direct regulation of the heart’s insulin receptor has not been explored. This study describes the first purification and characterization of the mammalian (rabbit, rat and bovine) heart insulin receptor. The rabbit heart IR showed maximum insulin binding of 18 μg/mg (~1 mole insulin/mole (α₂β₂) receptor) and a curvilinear Scatchard plot with a high affinity K_D for insulin binding of ~4 nM at optimal pH (7.8) and NaCl concentration (150 mM). The insulin receptor tyrosine kinase activity was stimulated by insulin, Mg²⁺ (half-maximum response at ~5.6–10.6 nM and ~8.5 mM, respectively) and by the physiological polyamines, spermine and spermidine. The stimulation by Mg²⁺ and the polyamines occurred with and without insulin. These characteristics of the heart insulin receptor provide a mechanism for regulating the activity of the receptor’s tyrosine kinase activity by the intracellular free Mg²⁺ concentration and the polyamines in the absence and presence of insulin.     

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca2+ concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca²⁺-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-β-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-β-cyclodextrin complexes restored the Ca²⁺-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca²⁺, annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca²⁺ concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca²⁺ concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

Ahsg-fetuin blocks the metabolic arm of insulin action through its interaction with the 95-kD β-subunit of the insulin receptor

We previously have shown that Ahsg, a liver glycoprotein, inhibits insulin receptor (InsR) tyrosine kinase (TK) activity and the ERK1/2 mitogenic signaling arm of insulin signaling. Here we show that Ahsg blocks insulin-stimulated GLUT4 translocation and Akt activation in intact cells (mouse myoblasts). Furthermore, Ahsg inhibits InsR autophosphorylation of highly-purified insulin holoreceptors in a cell-free, ATP-dependent system, with an IC₅₀ within the range of single-chain Ahsg concentrations in human serum. Binding of ¹²⁵I-insulin to living cells overexpressing the InsR shows a dissociation constant (K_D) of 250 pM, unaltered in the presence of 300 nM Ahsg. A mutant InsR cDNA encoding the signal peptide, the β-subunit and the furin processing site, but deleting the α-subunit, was stably expressed in HEK293 cells. Treatment with peroxovanadate, but not insulin, dramatically increased the 95 kD β-subunit tyrosine phosphoryation. The level of tyrosine phosphorylation of the 95-kD β-subunit can be driven down sharply by treatment of living HEK293 transfectant cells with physiological doses of Ahsg. Treatment of myogenic cells with Ahsg blunts insulin-stimulated InsR autophosphorylation and AKT phosphorylation. Taken together, we show that Ahsg antagonizes the metabolic functions initiated by InsR activation without interference in insulin binding. The experiments suggest a direct interaction of Ahsg with the InsR ectodomain β-subunit in a mode that does not significantly alter the high-affinity binding of insulin to the holoreceptor's two complementing α-subunits.     

Determining the target of membrane sterols on voltage-gated potassium channels

Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel K_v1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing K_v1.3 and K_v10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-β-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.     

Insulin Receptors and Downstream Substrates Associate with Membrane Microdomains after Treatment with Insulin or Chromium(III) Picolinate

We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)₃). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic)₃. These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)₃ but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)₃ did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)₃ likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)₃ involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)₃ are due to changes in membrane lipid order rather than to direct interactions with IR.     

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K⁺ and H⁺, and the organelle stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K⁺/H⁺ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MβCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K⁺ and H⁺. Loss of this membrane sterol is critical for the organelle acidification and stability.     

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit

The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.     

The role of cholesterol and sphingolipids in the dopamine D1 receptor and G protein distribution in the plasma membrane

G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein–lipid interactions play a pivotal role in cell signalling. Our previous results showed that although Gαs and Gαi₃ prefer different types of membrane domains they are both co-localized with the D₁ receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi₃ and their heterotrimers, as well as the D₁ receptor. We measured the lateral diffusion and membrane localization of investigated proteins using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by lifetime imaging microscopy (FLIM). The treatment with either methyl-β-cyclodextrin or Fumonisin B₁ led to the disruption of cholesterol–sphingolipids containing domains and changed the diffusion of Gαi₃ and the D₁ receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi₃ prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D₁ receptor exists in several different membrane localizations, depending on the receptor's conformation. We conclude that the inactive G protein heterotrimers are localized in the low-density membrane phase, from where they displace upon dissociation into the membrane-anchor- and subclass-specific lipid domain.     

Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-β-cyclodextrin (MβCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C_m), fluctuations of which reflect exocytosis and endocytosis. Treatment with MβCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca²⁺-evoked increase in C_m (IC₅₀ = 5.3 mM) vs. untreated cells. Cytosol dialysis of MβCD enhanced the attenuation of C_m increase (IC₅₀ = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MβCD resulted in an immediate C_m decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in C_m was three-fold slower than MβCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MβCD had no effect on the specific C_m and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MβCD-induced C_m decline. Thus acute exposure to MβCD evokes a C_m decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.     

Cholesterol-dependent regulation of adenosine A2A receptor-mediated anion secretion in colon epithelial cells

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. In this study we have investigated the role of cholesterol in the adenosine-dependent regulation of ion transport in colonic epithelial cells. We observed that methyl-β-cyclodextrin (MβCD), a cholesterol-sequestering molecule, enhanced adenosine A_2A receptor-activated transepithelial short circuit current (I_sc), but only from the basolateral side. Cholesterol is a major constituent of membrane microdomains, called lipid rafts that also contain sphingolipids. However, studies with the sphingomyelin-degrading enzyme, sphingomyelinase, and the cholesterol-binding agent, filipin, indicated that the change in the level of cholesterol alone was sufficient to control the adenosine-modulated I_sc. Cholesterol depletion had a major effect on the functional selectivity of A_2A receptors. Under control conditions, adenosine activated I_sc more potently than the specific A_2A agonist, CGS-21680, and the current was inhibited by XE991, an inhibitor of cAMP-dependent K⁺ channels. Following cholesterol depletion, CGS-21680 activated I_sc more potently than adenosine, and the current was inhibited by clotrimazole, an inhibitor of Ca²⁺-activated K⁺ (IK1) channels. Co-immunoprecipitation experiments revealed that A_2A receptors associate with IK1 channels following cholesterol depletion. These results suggest that cholesterol content in colonic epithelia affects adenosine-mediated anion secretion by controlling agonist-selective signaling.     

Membrane localization of Junín virus glycoproteins requires cholesterol and cholesterol rich membranes

Arenavirus morphogenesis and budding occurs at cellular plasma membrane; however, the nature of membrane assembly sites remains poorly understood. In this study we examined the effect of different cholesterol-lowering agents on Junín virus (JUNV) multiplication. We found that cholesterol cell depletion reduced JUNV glycoproteins (GPs) membrane expression and virus budding. Analysis of membrane protein insolubility in Triton X-100 suggested that JUNV GPs associate with cholesterol enriched membranes. Rafts dissociation conditions as warm detergent extraction and cholesterol removal by methyl-β-cyclodextrin compound showed to impair GPs cholesterol enriched membrane association. Analysis of GPs transfected cells showed similar results suggesting that membrane raft association is independent of other viral proteins.     

Membrane cholesterol content modulates activation of BK channels in colonic epithelia

Changes in the level of membrane cholesterol regulate a variety of signaling processes including those mediated by acylated signaling molecules that localize to lipid rafts. Recently several types of ion channels have been shown to have cholesterol-dependent activity and to localize to lipid rafts. In this study, we have investigated the role of cholesterol in the regulation of ion transport in colonic epithelial cells. We observed that methyl-β-cyclodextrin (MβCD), a cholesterol-sequestering molecule, activated transepithelial short circuit current (I_sc), but only from the basolateral side. Similar results were obtained with a cholesterol-binding agent, filipin, and with the sphingomyelin-degrading enzyme, sphingomyelinase. Experiments with ΔF508CFTR mutant mice indicated that raft disruption affected CFTR-mediated anion secretion, while pharmacological studies showed that this effect was due to activation of basolateral large conductance Ca²⁺-activated K⁺ (BK) channels. Sucrose density gradient centrifugation studies demonstrated that BK channels were normally present in the high-density fraction containing the detergent-insoluble cytoskeleton, and that following treatment with MβCD, BK channels redistributed into detergent-soluble fractions. Our evidence therefore implicates novel high-density cholesterol-enriched plasma membrane microdomains in the modulation of BK channel activation and anion secretion in colonic epithelia.     

A novel preparation of methyl-β-cyclodextrin from dimethyl carbonate and β-cyclodextrin

A novel green synthesis process about methyl-β-cyclodextrin has been investigated through the reaction between β-cyclodextrin and dimethyl carbonate by anhydrous potassium carbonate as catalyst in DMF. The influence of experimental factors including the molar ratio of dimethyl carbonate to β-cyclodextrin, reaction temperature, and reaction time on the average degree of substitution of methyl-β-cyclodextrin was studied. The results show that the average degree of substitution of methyl-β-cyclodextrin can be dependent on the reaction temperature and the molar ratio of raw material primarily. The structures of methyl-β-cyclodextrin were characterized by TLC, IR, MS, ¹H NMR, and ¹³C NMR.