Vm24, a natural immunosuppressive peptide, potently and selectively blocks Kv1.3 potassium channels of human T cells.

Blockade of Kv1.3 K(+) channels in T cells is a promising therapeutic approach for the treatment of autoimmune diseases such as multiple sclerosis and type 1 diabetes mellitus. Vm24 (α-KTx 23.1) is a novel 36-residue Kv1.3-specific peptide isolated from the venom of the scorpion Vaejovis mexicanus smithi. Vm24 inhibits Kv1.3 channels of human lymphocytes with high affinity (K(d) = 2.9 pM) and exhibits >1500-fold selectivity over other ion channels assayed. It inhibits the proliferation and Ca(2+) signaling of human T cells in vitro and reduces delayed-type hypersensitivity reactions in rats in vivo. Our results indicate that Vm24 has exceptional pharmacological properties that make it an excellent candidate for treatment of certain autoimmune diseases.

Developmental switch of the expression of ion channels in human dendritic cells.

Modulation of the expression and activity of plasma membrane ion channels is one of the mechanisms by which immune cells can regulate their intracellular Ca(2+) signaling pathways required for proliferation and/or differentiation. Voltage-gated K+ channels, inwardly rectifying K+ channels, and Ca(2+)-activated K+ channels have been described to play a major role in controlling the membrane potential in lymphocytes and professional APCs, such as monocytes, macrophages, and dendritic cells (DCs). Our study aimed at the characterization and identification of ion channels expressed in the course of human DC differentiation from monocytes. We report in this study for the first time that immature monocyte-derived DCs express voltage-gated Na+ channels in their plasma membrane. The analysis of the biophysical and pharmacological properties of the current and PCR-based cloning revealed the presence of Nav1.7 channels in immature DCs. Transition from the immature to a mature differentiation state, however, was accompanied by the down-regulation of Nav1.7 expression concomitant with the up-regulation of voltage-gated Kv1.3 K+ channel expression. The presence of Kv1.3 channels seems to be common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential.

Nutrition and immune system: certain fatty acids differently modify membrane composition and consequently kinetics of KV1.3 channels of human peripheral lymphocytes.

Potassium (K(+)) channels of human peripheral lymphocytes play a considerable role in the signalling processes required for immune responses. Modification of the fatty acid composition of the membrane influences the functions of various membrane enzymes and ion channels. We set out to establish how the incorporation of fatty acids with different carbon chain lengths and degrees of unsaturation into the cell membrane influences the function of K(V)1.3 channels of lymphocytes, thereby potentially modifying the immune responses of the cells. The incorporation of the fatty acids into the cell membrane was monitored by gas chromatography. Whole-cell patch-clamp experiments demonstrated that the polyunsaturated linoleic acid, arachidonic acid and docosahexaenoic acid all decreased the activation and inactivation time constants of the K(V)1.3 channels, but did not affect the voltage-dependence of steady-state activation and steady-state inactivation of the channels. Treatment with the saturated palmitic acid, stearic acid and the monounsaturated oleic acid did not result in significant changes in the biophysical parameters of K(V)1.3 gating studied. We conclude that the incorporation of fatty acids unsaturated to different degrees into the cell membrane of lymphocytes influenced the rate of gating transitions but not the equilibrium distribution of the channels between different states. This effect depended on the degree of unsaturation and the chain length of the fatty acids: no effects of saturated and monounsaturated fatty acids (16:0, 18:0 and 18:1) were observed whereas treatment with polyunsaturated fatty acids (18:2, 20:4 and 22:6) resulted in significant changes in the channel kinetics.

Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements.

Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Ralpha, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence.

Two novel toxins from the Amazonian scorpion Tityus cambridgei that block Kv1.3 and Shaker B K(+)-channels with distinctly different affinities.

Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4.

Novel alpha-KTx peptides from the venom of the scorpion Centruroides elegans selectively blockade Kv1.3 over IKCa1 K+ channels of T cells.

From the venom of the Mexican scorpion Centruroides elegans Thorell five peptides were isolated to homogeneity by chromatographic procedures and their full amino acid sequence was determined by automatic Edman degradation. They all belong to the Noxiustoxin subfamily of scorpion toxins and were given the systematic names alpha-KTx 2.8 to 2.12, with trivial names Ce1 to Ce5, respectively. They have 39 amino acid residues, except for Ce3 which has only 38, but all of them have three disulfide bridges, and have molecular weights of 4255, 4267, 4249, 4295 and 4255 atomic mass units, respectively for Ce1 to Ce5. The C-terminal residues of Ce2, Ce4 and Ce5 were found to be amidated. The electrophysiological assay (whole-cell patch-clamp) showed that out of the five peptides, Ce1 (alpha-KTx 2.8), Ce2 (alpha-KTX2.9) and Ce4 (alpha-KTx 2.11) were effective blockers of Kv1.3 channels of human T lymphocytes, whereas these peptides did not inhibit the Ca2+-activated K+ channels (IKCa1) of the same cells. The equilibrium dissociation constants of these peptides for Kv1.3 were 0.70, 0.25 and 0.98nM for Ce1, Ce2 and Ce4, respectively. Furthermore, toxins Ce1, Ce2 and Ce4 practically did not inhibit the related voltage gated Shaker K+ channels, and rKv2.1 channels of the Shab family. The high affinity blockage of Kv1.3 channels by these peptides and their selectivity for Kv1.3 over IKCa1 may have significance in the development of novel tools for suppressing the function of those T cell subsets whose proliferation critically depends on the activity of Kv1.3 channels.

Ion channels and lymphocyte activation.

The ion channels expressed by T lymphocytes play key roles in the control of the membrane potential and calcium signaling, thereby affecting signal transduction pathways that lead to the activation of these cells following antigenic stimulation. Disruption of these pathways can attenuate or prevent the response of T-cells to antigenic challenge resulting in immune suppression. Studies using ion channel blockers of high affinity and specificity have shown that this interference can be achieved at the level of ion channels. Suppression of immune functions by channel blockers has been demonstrated in vitro and in vivo. New information about the molecular structure of ion channels facilitates the design of more potent and more specific inhibitors. Thus, T-cell ion channels are likely to serve as targets for immunomodulatory drugs in the near future. Here, the biophysical properties, tissue distribution, regulation of expression, molecular pharmacology and role in T-cell activation of the voltage-gated Kv1.3 and the Ca(2+)-activated IKCa1 potassium channels and those of the Ca(+) release-activated Ca(2+) (CRAC) channel are reviewed.

Does mosaicism of the plasma membrane at molecular and higher hierarchical levels in human lymphocytes carry information on the immediate history of cells?

A theoretical analysis of experimental data is presented in this mini-review on non-random homo- and hetero-associations of cell surface receptors, which can be recruited in the plasma membrane or at the surface of the rough endoplasmic reticulum during the protein synthesis. In the latter case, the likely genetic origin of these supramolecular formations is analyzed, contrasting this concept to the mobility of the cell surface proteins. A model is offered which, on the one hand, allows the mobility in a restricted way even among microdomain-confined receptor proteins through 'swapping partners'. On the other hand, the lack of mixing molecular components of protein clusters will be analyzed, when homo-and hetero-associations are studied through cell fusion experiments. The most frequently studied cell surface patterns have included lipid raft organized HLA class I and II, ICAM-1, tetraspan molecules, IL2 and IL15 and other receptors, as well. On the contrary coated pit-associated transferrin receptors would not mix with the above lipid raft associated receptor patterns, although transferrin receptor would readily oligomerize into homo-associates. The functional consequences of these superstructures are also analyzed. On the 30th anniversary of the Singer-Nicolson fluid mosaic membrane model one has to pay tribute to the authors, because of their deep insight emphasizing also the mosaicism of the membranes in general and that of the plasma membrane, in particular.

Signal transduction in T lymphocytes and aging.

Subclasses of cells in different compartments of the immune system possesses all those attributes, that make them suitable though somewhat limited models for the investigation of cellular processes during aging. Blood samples provide relative easily high amount of cells belonging to the same subclass, all of them having complex cascade processes in their signal transduction mechanisms, therefore being excellent targets for such investigations. One such subclass comprises peripheral blood lymphocytes. The signal-transduction cascade across the plasma membrane of lymphocytes displays many of the general features enabling us to draw conclusions for other cellular signaling problems that may arise during aging in other cell types not directly related to the immune system. The advantage of this approach lies in the fact that sometimes it is extremely difficult to study signal transduction processes in certain cell types under physiological conditions. The simultaneous occurrence of physical, chemical and molecular biological regulation of the immune processes at cellular and network levels make them very good examples for focusing our interest also on similar processes in other systems and cells. The fast developing new measuring techniques and the rapidly accumulating experimental data make it relatively easy to provide interesting new aspects, and ideas in this field. Finally, the immune system itself has its great importance and after all, it has an obvious declination with aging, the immune-senescence.

Computer program for determining fluorescence resonance energy transfer efficiency from flow cytometric data on a cell-by-cell basis.

The determination of fluorescence resonance energy transfer (FRET) with flow cytometry (FCET) is one of the most efficient tools to study the proximity relationships of cell membrane components in cell populations on a cell-by-cell basis. Because of the high amount of data and the relatively tedious calculations, this procedure should be assisted by powerful data processing software. The currently available programs are not able to fulfill this requirement. We developed a Windows-based program to calculate fluorescence resonance energy transfer efficiency values from list mode flow cytometry standard (FCS) files. This program displays the measured data in standard plots by generating one- and two-parameter histograms on linear or logarithmic scales. A graphical gating tool allows the user to select the desired cell population according to any combination of the parameter values. The program performs several statistical calculations, including mean, S.D., percent of the gated data. We have implemented two types of data sheet for FRET calculations to aid and guide the user during the analysis: one with population-mean-based autofluorescence correction and the other with spectrum-based cell-by-cell autofluorescence correction. In this paper, we describe the gating algorithms, the file opening procedure and the rules of gating. The structure of the program and a short description of the graphical user-interface (GUI) are also presented in this article.

Tst26, a novel peptide blocker of Kv1.2 and Kv1.3 channels from the venom of Tityus stigmurus.

Using high-performance liquid chromatography Tst26, a novel potassium channel blocker peptide, was purified from the venom of the Brazilian scorpion Tityus stigmurus. Its primary structure was determined by means of automatic Edman degradation and mass spectrometry analysis. The peptide is composed of 37 amino acid residues and tightly folded through three disulfide bridges, similar to other K(+) channel blocking peptides purified from scorpion venoms. It contains the "essential dyad" for K(+) channel recognition comprised of a lysine at position 27 and a tyrosine at position 36. Electrophysiological assays revealed that Tst26 blocked hKv1.2 and hKv1.3 channels with high affinity (K(d)=1.9 nM and 10.7 nM, respectively) while it did not affect several other ion channels (mKv1.1, hKv1.4, hKv1.5, hERG, hIKCa1, hBK, hNav1.5) tested at 10 nM concentration. The voltage-dependent steady-state parameters of K(+) channel gating were unaffected by the toxin in both channels, but due to the fast association and dissociation kinetics Tst26 slowed the rate of inactivation of Kv1.3 channels. Based on the primary structure, the systematic nomenclature proposed for this peptide is alpha-KTx 4.6.

Effects of changes in extracellular pH and potassium concentration on Kv1.3 inactivation.

The Kv1.3 channel inactivates via the P/C-type mechanism, which is influenced by a histidine residue in the pore region (H399, equivalent of Shaker 449). Previously we showed that the electric field of the protonated histidines at low extracellular pH (pHe) creates a potential barrier for K+ ions just outside the pore that hinders their exit from the binding site controlling inactivation (control site) thereby slowing inactivation kinetics. Here we examined the effects of extracellular potassium [K+]e and pHe on the rate of inactivation of Kv1.3 using whole-cell patch-clamp. We found that in 150 mM [K+]e inactivation was accelerated upon switching to pHe 5.5 as opposed to the slowing at 5 mM [K+]e. The transition from slowing to acceleration occurred at 40 mM [K+]e, whereas this "turning point" was at 20 mM [K+]e for inward currents. The rate of entry of Ba(2+) ions from the extracellular space to the control site was significantly slowed by low pHe in wild-type hKv1.3, but it was insensitive to pH(e) in H399K and H399L mutants. Based on these observations we expanded our model and propose that the potential barrier created by the protonated histidines impedes the passage of K+ ions between the extracellular medium and the control site in both directions and the effect on inactivation rate (acceleration or slowing) depends on the relative contribution of filling from the extracellular and intracellular sides.

A selective blocker of Kv1.2 and Kv1.3 potassium channels from the venom of the scorpion Centruroides suffusus suffusus.

A novel potassium channel blocker peptide was purified from the venom of the scorpion Centruroides suffusus suffusus by high-performance liquid chromatography and its amino acid sequence was completed by Edman degradation and mass spectrometry analysis. It contains 38 amino acid residues with a molecular weight of 4000.3Da, tightly folded by three disulfide bridges. This peptide, named Css20, was shown to block preferentially the currents of the voltage-dependent K+-channels Kv1.2 and Kv1.3. It did not affect several other ion channels tested at 10 nM concentration. Concentration-response curves of Css20 yielded an IC50 of 1.3 and 7.2 nM for Kv1.2- and Kv1.3-channels, respectively. Interestingly, despite the similar affinities for the two channels the association and dissociation rates of the toxin were much slower for Kv1.2, implying that different interactions may be involved in binding to the two channel types; an implication further supported by in silico docking analyses. Based on the primary structure of Css20, the systematic nomenclature proposed for this toxin is alpha-KTx 2.13.

Involvement of membrane channels in autoimmune disorders.

Ion channels are ubiquitous transmembrane proteins that are involved in a wide variety of cellular functions by selectively controlling the passage of ions across the plasma membrane. Among these functions many immune processes, including those in autoimmune reactions, also rely on the operation of ion channels, but the roles of ion channels can be very diverse. Here the participation of ion channels in three different roles in autoimmune processes is discussed: 1. ion channels in effector immune cells attacking other tissues causing autoimmune diseases, such as multiple sclerosis; 2. ion channels as direct targets of the immune system whereby loss of channel function leads to disease, as in myasthenia gravis; 3. ion channels whose function is modulated in the target cells by an apoptotic signal transduction cascade, such as the Fas/Fas ligand pathway. The numerous tasks that ion channels perform in autoimmune disorders and the wealth of information that has been gathered about them in recent years together provide a good basis for the design and production of drugs that may be effectively used in the therapy of these diseases.

Detection of channel proximity by nanoparticle-assisted delaying of toxin binding; a combined patch-clamp and flow cytometric energy transfer study.

Gold nanoparticles of 30 nm diameter bound to cell-surface receptor major histocompatibility complex glycoproteins (MHCI and MHCII), interleukin-2 receptor alpha subunit (IL-2Ralpha), very late antigen-4 (VLA-4) integrin, transferrin receptor, and the receptor-type protein tyrosin phosphatase CD45 are shown by the patch-clamp technique to selectively modulate binding characteristics of Pi(2) toxin, an efficient blocker of K(v)1.3 channels. After correlating the electrophysiological data with those on the underlying receptor clusters obtained by simultaneously conducted flow cytometric energy transfer measurements, the modulation was proved to be sensitive to the density and size of the receptor clusters, and to the locations of the receptors as well. Based on the observation that engagement of MHCII by a monoclonal antibody down-regulates channel current and based on the close nanometer-scale proximity of the MHCI and MHCII glycoproteins, an analogous experiment was carried out when gold nanoparticles bound to MHCI delayed down-regulation of the K(v)1.3 current initiated by ligation of MHCII. Localization of K(v)1.3 channels in the nanometer-scale vicinity of the MHC-containing lipid rafts is demonstrated for the first time. A method is proposed for detecting receptor-channel or receptor-receptor proximity by observing nanoparticle-induced increase in relaxation times following concentration jumps of ligands binding to channels or to receptors capable of regulating channel currents.

Nanoparticle energy transfer on the cell surface.

Membrane topology of receptors plays an important role in shaping transmembrane signalling of cells. Among the methods used for characterizing receptor clusters, fluorescence resonance energy transfer between a donor and acceptor fluorophore plays a unique role based on its capability of detecting molecular level (2-10 nm) proximities of receptors in physiological conditions. Recent development of biotechnology has made possible the usage of colloidal gold particles in a large size range for specific labelling of cells for the purposes of electron microscopy. However, by combining metal and fluorophore labelling of cells, the versatility of metal-fluorophore interactions opens the way for new applications by detecting the presence of the metal particles by the methods of fluorescence spectroscopy. An outstanding feature of the metal nanoparticle-fluorophore interaction is that the metal particle can enhance spontaneous emission of the fluorophore in a distance-dependent fashion, in an interaction range essentially determined by the size of the nanoparticle. In our work enhanced fluorescence of rhodamine and cyanine dyes was observed in the vicinity of immunogold nanoparticles on the surface of JY cells in a flow cytometer. The dyes and the immunogold were targetted to the cell surface receptors MHCI, MHCII, transferrin receptor and CD45 by monoclonal antibodies. The fluorescence enhancement was sensitive to the wavelength of the exciting light, the size and amount of surface bound gold beads, as well as the fluorophore-nanoparticle distance. The intensity of 90 degrees scattering of the incident light beam was enhanced by the immunogold in a concentration and size-dependent fashion. The 90 degrees light scattering varied with the wavelength of the incident light in a manner characteristic to gold nanoparticles of the applied sizes. A reduction in photobleaching time constant of the cyanine dye was observed in the vicinity of gold particles in a digital imaging microscope. Modulations of 90 degrees light scattering intensity and photobleaching time constant indicate the role of the local field in the fluorescence enhancement. A mathematical simulation based on the electrodynamic theory of fluorescence enhancement showed a consistency between the measured enhancement values, the inter-epitope distances and the quantum yields. The feasibility of realizing proximity sensors operating at distance ranges larger than that of the conventional Forster transfer is demonstrated on the surface of living cells.

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

Anuroctoxin, a new scorpion toxin of the alpha-KTx 6 subfamily, is highly selective for Kv1.3 over IKCa1 ion channels of human T lymphocytes.

The physiological function of T lymphocytes can be modulated selectively by peptide toxins acting on Kv1.3 K(+) channels. Because Kv1.3-specific peptide toxins are considered to have a significant therapeutic potential in the treatment of autoimmune diseases, the discovery of new toxins is highly motivated. Through chromatographic procedures and electrophysiological assays, using patch-clamp methodology, the isolation of a novel peptide named anuroctoxin was accomplished using the venom of the Mexican scorpion Anuroctonus phaiodactylus. It has 35 amino acid residues with a molecular weight of 4082.8, tightly bound by four disulfide bridges whose complete covalent structure was determined. It has a pyroglutamic acid at the N-terminal region and an amidated C-terminal residue. Sequence comparison and phylogenetic clustering analysis classifies anuroctoxin into subfamily 6 of the alpha-KTx scorpion toxins (systematic name, alpha-KTx 6.12). Patch-clamp experiments show that anuroctoxin is a high-affinity blocker of Kv1.3 channels of human T lymphocytes with a K(d) of 0.73 nM, and it does not block the Ca(2+)-activated IKCa1 K(+) channels. These two channels play different but important roles in T-lymphocyte activation. Furthermore, the toxin practically does not inhibit Shaker IR, mKv1.1, and rKv2.1 channels, whereas the affinity of anuroctoxin for hKv1.2 is almost an order of magnitude smaller than for Kv1.3. The pharmacological profile and the selectivity of this new toxin for Kv1.3 over IKCa1 may provide an important tool for the modulation of the immune system, especially in cases in which selective inhibition of Kv1.3 is required.

Active and passive behaviour in the regulation of stiffness of the lateral wall in outer hair cells of the guinea-pig.

The stiffness of the outer hair cell (OHC) lateral wall, measured by the micropipette aspiration technique, is non-linear, decreasing from the ciliary pole (stiffness parameter Sp 1.83+/-0.13 nN/microm n=10) towards the cell base (Sp 1.14+/-0.16 nN/microm, n=10) irrespective of the cochleoapical or cochleobasal origin of the cells. The length of the aspirated lateral wall segment was related exponentially to the duration of the applied negative pressure (6 cm H2O) in the synaptic region of the OHCs whereas an active, sigmoid component was observed between 30 and 60 s in the supranuclear regions. A significant increase of the midlateral wall stiffness (to 1.91+/-0.23 nN/microm; n=10) was observed in calcium-free medium and the sigmoid component of the response of the lateral wall was abolished. Salicylate (5 mM) had no significant effect on the active sigmoid behaviour of the lateral wall (n=10). Gadolinium (5 mM), a non-specific cation channel blocker, increased the stiffness of the lateral wall and attenuated the active component (n=10). The motor protein prestin thus does not seem to be involved in the active stiffness regulation seen in this study. A role for the cortical cytoskeleton in the regulation of stiffness seems reasonable according to our model. The mechanism may involve calcium-dependent metabolic modification of cytoskeletal or membrane proteins.

Cholesterol modifies the gating of Kv1.3 in human T lymphocytes.

The Kv1.3 potassium channel that belongs to the Shaker family of voltage-gated K(+) channels plays a crucial role in the mitogenic response of T cells. Because it spans the cell membrane its function can be influenced by lipid-protein interactions. In order to study the effect of lipid-protein interactions on the functioning of Kv1.3 we manipulated the membrane cholesterol content in T cells mimicking various physiological conditions by means of the oligosaccharide methyl-beta-cyclodextrin (MbetaCD) and its cholesterol-saturated complex (MbetaCD/C). Fluorescence polarization anisotropy and peak current density were used to monitor the efficiency of cholesterol removal (MbetaCD) and loading (MbetaCD/C). Using whole-cell patch-clamp technique we determined the kinetic and steady-state parameters of activation and inactivation of the Kv1.3 currents under different treatment conditions. Upon elevation of cholesterol content by 1 or 1.5 mg/ml MbetaCD/C the rates of both activation and inactivation were slowed. Moreover, the increased cholesterol level in the membrane resulted in a biphasic activation curve. Cholesterol depletion with MbetaCD (0.95 and 1.425 mg/ml) caused no significant changes in the gating characteristics of Kv1.3. The equilibrium between the open and the closed states of the channels was affected by increased cholesterol content, but at the same time steady-state inactivation was unchanged. We argue that manipulation of membrane cholesterol changed both the kinetic properties of Kv1.3 and steady-state parameters of activation by modifying lipid-protein interactions.