RESUMEN
Delayed-rectifier K+ currents (I(DR)) in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltage-gated K+ channel, K(V)2.1, is expressed in beta-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I(DR) in rodent beta-cells. A novel peptidyl inhibitor of K(V)2.1/K(V)2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration approximately 1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to beta-cell physiology. In mouse beta-cells, GxTX-1 inhibits 90% of I(DR) and, as for K(V)2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the beta-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the beta-cell I(DR), which may provide advantages over currently used therapies for the treatment of type 2 diabetes.
Asunto(s)
Canales de Potasio de Tipo Rectificador Tardío/fisiología , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Secuencia de Aminoácidos , Animales , Canales de Potasio de Tipo Rectificador Tardío/efectos de los fármacos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/química , Venenos de Araña/química , Venenos de Araña/farmacologíaRESUMEN
The Trans Cell Layer Electrical Field Stimulation (TCL-EFS) system has been developed for high-throughput screening (HTS) of voltage-gated ion channels in microplate format on a Voltage-Ion Probe Reader (VIPR) platform. In this design, a wire electrode is placed above the cell layer of each filter well, and a whole plate perimeter electrode resides beneath the filter layer. This configuration allows the electrodes to be placed away from the cell layer to minimize the near electrode field effects on cell function and dye bleaching observed with other existing designs. Mathematical simulation indicates that the electric field at the cell layer becomes uniform as the top electrode is raised to a position near the surface of the solution in the well. Using the TCL-EFS system and membrane potential fluorescence resonance energy transfer (FRET) dyes, the sensitivity of voltage-gated sodium channels to tetrodotoxin and other channel inhibitors was found to be similar to those determined by established electrophysiological and more conventional VIPR techniques. A good correlation was also observed with the TCL-EFS system for inhibition of Cav2.2 by omega-conotoxin-GVIA and for block of Cav1.2 by known small molecule inhibitors. Thus, the TCLEFS system is suitable for both quantitative analysis and HTS of voltage-gated sodium and calcium channels, without the liabilities of previously reported EFS methodologies.
Asunto(s)
Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Proteínas Musculares/fisiología , Canales de Sodio/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Simulación por Computador , Estimulación Eléctrica , Electrofisiología/instrumentación , Electrofisiología/métodos , Humanos , Cinética , Proteínas Musculares/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5 , Canales de Sodio/efectos de los fármacos , Tetrodotoxina/farmacología , omega-Conotoxina GVIA/farmacologíaRESUMEN
Clinical treatment of neuropathic pain can be achieved with a number of different drugs, some of which interact with all members of the voltage-gated sodium channel (NaV1) family. However, block of central nervous system and cardiac NaV1 channels can cause dose-limiting side effects, preventing many patients from achieving adequate pain relief. Expression of the tetrodotoxin-resistant NaV1.8 subtype is restricted to small-diameter sensory neurons, and several lines of evidence indicate a role for NaV1.8 in pain processing. Given these features, NaV1.8 subtype-selective blockers are predicted to be efficacious in the treatment of neuropathic pain and to be associated with fewer adverse effects than currently available therapies. To facilitate the identification of NaV1.8-specific inhibitors, we stably expressed the human NaV1.8 channel together with the auxiliary human beta1 subunit (NaV beta1) in human embryonic kidney 293 cells. Heterologously expressed human NaV1.8/NaV beta1 channels display biophysical properties that are similar to those of tetrodotoxin-resistant channels present in mouse dorsal root ganglion neurons. A membrane potential, fluorescence resonance energy transfer-based functional assay on a fluorometric imaging plate reader (FLIPR-Tetra, Molecular Devices, Sunnyvale, CA) platform has been established. This highcapacity assay is sensitive to known state-dependent NaV1 modulators and can be used to identify novel and selective NaV1.8 inhibitors.
Asunto(s)
Potenciales de la Membrana/fisiología , Neuronas Aferentes/fisiología , Canales de Sodio/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN , Electrofisiología/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Riñón , Modelos Moleculares , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.8 , Fragmentos de Péptidos/inmunología , Conformación Proteica , Conejos , Canales de Sodio/genéticaRESUMEN
Voltage-gated potassium channels (Kv channels) are involved in repolarization of excitable cells. In pancreatic beta-cells, prolongation of the action potential by block of delayed rectifier potassium channels would be expected to increase intracellular free calcium and to promote insulin release in a glucose-dependent manner. However, the specific Kv channel subtypes responsible for repolarization in beta-cells, most importantly in humans, are not completely resolved. In this study, we have investigated the expression of 26 subtypes from Kv subfamilies in human islet mRNA. The results of the RT-PCR analysis were extended by in situ hybridization and/or immunohistochemical analysis on sections from human or Rhesus pancreas. Cell-specific markers were used to show that Kv2.1, Kv3.2, Kv6.2, and Kv9.3 are expressed in beta-cells, that Kv3.1 and Kv6.1 are expressed in alpha-cells, and that Kv2.2 is expressed in delta-cells. This study suggests that more than one Kv channel subtype might contribute to the beta-cell delayed rectifier current and that this current could be formed by heterotetramers of active and silent subunits.
Asunto(s)
Islotes Pancreáticos/fisiología , Canales de Potasio/genética , Animales , Secuencia de Bases , Cartilla de ADN , Regulación de la Expresión Génica , Humanos , Islotes Pancreáticos/citología , Macaca mulatta , Canales de Potasio/clasificación , ARN Mensajero/genética , Canales de Potasio Shab , Especificidad de la EspecieRESUMEN
The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.
Asunto(s)
Colorantes/análisis , Transferencia Resonante de Energía de Fluorescencia/métodos , Canales de Sodio/análisis , Canales de Sodio/fisiología , Línea Celular , Colorantes/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas Musculares/análisis , Proteínas Musculares/fisiología , Canal de Sodio Activado por Voltaje NAV1.5 , Canal de Sodio Activado por Voltaje NAV1.7 , Bloqueadores de los Canales de Sodio/farmacología , Veratridina/farmacologíaRESUMEN
Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well-characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. Chinese hamster ovary (CHO)-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. Stichodactyla helianthus peptide (ShK), isolated from S. helianthus venom and a known high-affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit 86Rb+ efflux from CHO-K1.hKv3.2b (IC50 approximately 0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus laevis oocytes or in planar patch-clamp studies, ShK inhibited hKv3.2b channels with IC50 values of approximately 0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic beta cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with the blockade of Kv3 channels (i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential after hyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections). Taken together, these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations.
Asunto(s)
Venenos de Cnidarios/farmacología , Fragmentos de Péptidos/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Canales de Potasio con Entrada de Voltaje/biosíntesis , Animales , Células CHO , Venenos de Cnidarios/aislamiento & purificación , Cricetinae , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Fragmentos de Péptidos/aislamiento & purificación , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Bloqueadores de los Canales de Potasio/farmacología , Anémonas de Mar , Canales de Potasio ShawRESUMEN
Kv1.3, the voltage-gated potassium channel in human T cells, represents a new target for treating immunosuppression and autoimmune diseases. Correolide (1), a pentacyclic natural product, is a potent and selective Kv1.3 channel blocker. Simplification of correolide via removal of its E-ring generates enone 4, whose modification produced a new series of tetracyclic Kv1.3 blockers. The structure-activity relationship for this class of compounds in two functional assays, Rb_Kv and human T cell proliferation, is presented herein. The most potent analog 43 is 15-fold more potent than correolide as inhibitor of human T cell proliferation.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Triterpenos/farmacología , Bioensayo , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Canal de Potasio Kv1.3 , Modelos Moleculares , Bloqueadores de los Canales de Potasio/química , Relación Estructura-Actividad , Linfocitos T , Triterpenos/químicaRESUMEN
Di-substituted cyclohexyl (DSC) derivatives inhibit the voltage-gated potassium channel, K(v)1.3, and have immunosuppressant activity (Schmalhofer et al. (2002) Biochemistry 41, 7781-7794). This class of inhibitors displays Hill coefficients of near 2 in functional assays, and trans DSC analogues appear to selectively interact with K(v)1.3 channel conformations related to C-type inactivation. To further understand the details of the DSC inhibitor interaction with potassium channels, trans-1-(N-n-propylcarbamoyloxy)-4-phenyl-4-(3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl)cyclo-hexane (trans-NPCO-DSC) was radiolabeled with tritium, and its binding characteristics to K(v)1.3 channels were determined. Specific binding of [(3)H]-trans-NPCO-DSC to K(v)1.3 channels is a saturable, time-dependent, and fully reversible process. Saturation binding isotherms and competition binding experiments are consistent with the presence of two receptor sites for DSC derivatives on the K(v)1.3 channel that display positive allosteric cooperativity. The high affinity interaction of [(3)H]-trans-NPCO-DSC with K(v)1.3 channels appears to correlate with the rates of C-type inactivation of the channel. These data, taken together, mark the first demonstration of the existence of multiple binding sites for an inhibitor of an ion channel and suggest that the high affinity interaction of trans-NPCO-DSC and similar inhibitors with K(v)1.3 channels could be exploited for the development of selective molecules that target this protein.
Asunto(s)
Compuestos Aza/metabolismo , Ciclohexanos/metabolismo , Bloqueadores de los Canales de Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Compuestos Aza/química , Sitios de Unión , Unión Competitiva , Ciclohexanos/química , Isomerismo , Cinética , Canal de Potasio Kv1.3 , Bloqueadores de los Canales de Potasio/química , Unión ProteicaRESUMEN
ShK, a peptide isolated from Stichodactyla helianthus venom, blocks the voltage-gated potassium channels, K(v)1.1 and K(v)1.3, with similar high affinity. ShK-Dap(22), a synthetic derivative in which a diaminopropionic acid residue has been substituted at position Lys(22), has been reported to be a selective K(v)1.3 inhibitor and to block this channel with equivalent potency as ShK [Kalman et al. (1998) J. Biol. Chem. 273, 32697-32707]. In this study, a large body of evidence is presented which indicates that the potencies of wild-type ShK peptide for both K(v)1.3 and K(v)1.1 channels have been previously underestimated. Therefore, the affinity of ShK-Dap(22) for both channels appears to be ca. 10(2)-10(4)-fold weaker than ShK. ShK-Dap(22) does display ca. 20-fold selectivity for human K(v)1.3 vs K(v)1.1 when measured by the whole-cell voltage clamp method but not in equilibrium binding assays. ShK-Dap(22) has low affinity for K(v)1.2 channels, but heteromultimeric K(v)1.1-K(v)1.2 channels form a receptor with ca. 200-fold higher affinity for ShK-Dap(22) than K(v)1.1 homomultimers. In fact, K(v)1.1-K(v)1.2 channels bind ShK-Dap(22) with only ca. 10-fold less potency than ShK and reveal a novel pharmacology not predicted from the homomultimers of K(v)1.1 or K(v)1.2. The concentrations of ShK-Dap(22) needed to inhibit human T cell activation were ca. 10(3)-fold higher than those of ShK, in good correlation with the relative affinities of these peptides for inhibiting K(v)1.3 channels. All of these data, taken together, suggest that ShK-Dap(22) will not have the same in vivo immunosuppressant efficacy of other K(v)1.3 blockers, such as margatoxin or ShK. Moreover, ShK-Dap(22) may have undesired side effects due to its interaction with heteromultimeric K(v)1.1-K(v)1.2 channels, such as those present in brain and/or peripheral tissues.
Asunto(s)
Venenos de Cnidarios/química , Venenos de Cnidarios/farmacología , Péptidos/química , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Sustitución de Aminoácidos , Animales , Encéfalo/metabolismo , Células CHO , Línea Celular , Venenos de Cnidarios/genética , Cricetinae , Humanos , Concentración 50 Inhibidora , Potenciales de la Membrana/efectos de los fármacos , Oocitos/metabolismo , Péptidos/genética , Bloqueadores de los Canales de Potasio/química , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Ensayo de Unión Radioligante , Anémonas de Mar/química , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , XenopusRESUMEN
The voltage-gated potassium channel, K(v)1.3, is a novel target for development of immunosuppressants. Using a functional (86)Rb(+) efflux assay, a new class of high-affinity K(v)1.3 inhibitors has been identified. The initial active in this series, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone (PAC), which is representative of a disubstituted cyclohexyl (DSC) template, displays a K(i) of ca. 300 nM and a Hill coefficient near 2 in the flux assay and in voltage clamp recordings of K(v)1.3 channels in human T-lymphocytes. PAC displays excellent specificity as it only blocks members of the K(v)1 family of potassium channels but does not affect many other types of ion channels, receptors, or enzyme systems. Block of K(v)1.3 by DSC analogues occurs with a well-defined structure-activity relationship. Substitution at the C-1 ketone of PAC generates trans (down) and cis (up) isomer pairs. Whereas many DSC derivatives do not display selectivity in their interaction with different K(v)1.x channels, trans DSC derivatives distinguish between K(v)1.x channels based on their rates of C-type inactivation. DSC analogues reversibly inhibit the Ca(2+)-dependent pathway of T cell activation in in vitro assays. Together, these data suggest that DSC derivatives represent a new class of immunosuppressant agents and that specific interactions of trans DSC analogues with channel conformations related to C-type inactivation may permit development of selective K(v)1.3 channel inhibitors useful for the safe treatment of autoimmune diseases.
Asunto(s)
Ciclohexanonas/farmacología , Inmunosupresores/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Alanina/genética , Animales , Sitios de Unión , Células CHO , Línea Celular , Cricetinae , Ciclohexanonas/síntesis química , Ciclohexanonas/metabolismo , Cobayas , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/metabolismo , Líquido Intracelular/metabolismo , Canal de Potasio Kv1.3 , Activación de Linfocitos/efectos de los fármacos , Monoyodotirosina/metabolismo , Técnicas de Placa-Clamp , Fenilalanina/genética , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Ratas , Venenos de Escorpión/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transfección , Triterpenos/metabolismo , Tirosina/genéticaRESUMEN
The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.