T淋巴细胞离子通道的研究进展

研究表明,离子通道CRAC、Kv1.3、KCa3.1、Clswell等共同在T淋巴细胞活化及稳态中发挥作用,参与机体免疫调节.钙离子通过T淋巴细胞膜上的CRAC进入细胞内,作为第二信使激活T淋巴细胞.钾离子通道介导的钾离子外流是T淋巴细胞膜电位形成的基础,间接调节T淋巴细胞的活化和功能.T淋巴细胞膜上的氯离子通道与细胞的体积调节有关.了解离子通道表达改变所引起T细胞亚型的分化,T细胞离子通道在T细胞活化中的作用及离子通道阻断剂用于治疗自身免疫疾病及过敏反应的可能性具有重要意义.     

Ion channels and their functional role in vascular endothelium

Endothelial cells (EC) form a unique signal-transducing surface in the vascular system. The abundance of ion channels in the plasma membrane of these nonexcitable cells has raised questions about their functional role. This review presents evidence for the involvement of ion channels in endothelial cell functions controlled by intracellular Ca(2+) signals, such as the production and release of many vasoactive factors, e.g., nitric oxide and PGI(2). In addition, ion channels may be involved in the regulation of the traffic of macromolecules by endocytosis, transcytosis, the biosynthetic-secretory pathway, and exocytosis, e.g., tissue factor pathway inhibitor, von Willebrand factor, and tissue plasminogen activator. Ion channels are also involved in controlling intercellular permeability, EC proliferation, and angiogenesis. These functions are supported or triggered via ion channels, which either provide Ca(2+)-entry pathways or stabilize the driving force for Ca(2+) influx through these pathways. These Ca(2+)-entry pathways comprise agonist-activated nonselective Ca(2+)-permeable cation channels, cyclic nucleotide-activated nonselective cation channels, and store-operated Ca(2+) channels or capacitative Ca(2+) entry. At least some of these channels appear to be expressed by genes of the trp family. The driving force for Ca(2+) entry is mainly controlled by large-conductance Ca(2+)-dependent BK(Ca) channels (slo), inwardly rectifying K(+) channels (Kir2.1), and at least two types of Cl( -) channels, i.e., the Ca(2+)-activated Cl(-) channel and the housekeeping, volume-regulated anion channel (VRAC). In addition to their essential function in Ca(2+) signaling, VRAC channels are multifunctional, operate as a transport pathway for amino acids and organic osmolytes, and are possibly involved in endothelial cell proliferation and angiogenesis. Finally, we have also highlighted the role of ion channels as mechanosensors in EC. Plasmalemmal ion channels may signal rapid changes in hemodynamic forces, such as shear stress and biaxial tensile stress, but also changes in cell shape and cell volume to the cytoskeleton and the intracellular machinery for metabolite traffic and gene expression.     

M(1) and M(2) muscarinic acetylcholine receptor subtypes mediate Ca(2+) channel current inhibition in rat sympathetic stellate ganglion neurons

Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca(2+) channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca(2+) channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca(2+) channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca(2+) channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M-mediated mAChR stimulation led to inhibition of Ca(2+) currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M(1) receptor blocker, M(1)-toxin, resulted in VD inhibition of Ca(2+) currents after Oxo-M application. On the other hand, VI modulation of Ca(2+) currents was observed after pretreatment of cells with methoctramine (M(2) mAChR blocker). The Oxo-M-mediated inhibition was nearly eliminated in the presence of both M(1) and M(2) mAChR blockers but was unaltered when SG neurons were exposed to the M(4) mAChR toxin, M(4)-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M(1) and M(2) receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M(1) and M(2) mAChR, and activation of these receptors leads to inhibition of Ca(2+) channel currents through VI and VD pathways, respectively.     

BK channel openers inhibit migration of human glioma cells

Large-conductance Ca(2+)-activated K(+) channels (BK channels) are highly expressed in human glioma cells. However, less is known about their biological function in these cells. We used the patch-clamp technique to investigate activation properties of BK channels and time-lapse microscopy to evaluate the role of BK channel activation in migration of 1321N1 human glioma cells. In whole cells, internal perfusion with a solution containing 500 nM free Ca(2+) and external application of the BK channel opener phloretin (100 micro M) shifted the activation threshold of BK channel currents toward more negative voltages of about -30 mV, which is close to the resting potential of the cells. The concentration of intracellular Ca(2+) in fura-2-loaded 1321N1 cells was measured to be 235+/-19 nM and was increased to 472+/-25 nM after treatment with phloretin. Phloretin and another BK channel opener NS1619 (100 micro M) reduced the migration velocity by about 50%. A similar reduction was observed following muscarinic stimulation of glioma cells with acetylcholine (100 micro M). The effects of phloretin, NS1619 and acetylcholine on cell migration were completely abolished by co-application of the specific BK channel blockers paxilline (5 micro M) and iberiotoxin (100 nM). The phloretin-induced increase in intracellular Ca(2+) was unaffected by the removal of extracellular Ca(2+) and co-application of paxilline. These findings indicate that glioma cell migration was inhibited through BK channel activation, independent of intracellular Ca(2+).     

Low-threshold L-type calcium channels in rat dopamine neurons

Ca(2+) channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 microM), omega-conotoxin GVIA (Ctx, 1 microM), or omega-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca(2+) channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca(2+) channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca(2+) channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca(2+)-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca(2+) channels may exhibit different thresholds for activation in different types of neurons.     

Permeation properties of the hair cell mechanotransducer channel provide insight into its molecular structure

Mechanoelectric transducer (MET) channels, located near stereocilia tips, are opened by deflecting the hair bundle of sensory hair cells. Defects in this process result in deafness. Despite this critical function, the molecular identity of MET channels remains a mystery. Inherent channel properties, particularly those associated with permeation, provide the backbone for the molecular identification of ion channels. Here, a novel channel rectification mechanism is identified, resulting in a reduced pore size at positive potentials. The apparent difference in pore dimensions results from Ca(2+) binding within the pore, occluding permeation. Driving force for permeation at hyperpolarized potentials is increased because Ca(2+) can more easily be removed from binding within the pore due to the presence of an electronegative external vestibule that dehydrates and concentrates permeating ions. Alterations in Ca(2+) binding may underlie tonotopic and Ca(2+)-dependent variations in channel conductance. This Ca(2+)-dependent rectification provides targets for identifying the molecular components of the MET channel.     

The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis

The transport of calcium ions (Ca(2+)) to the cytosol is essential for immunoreceptor signaling, regulating lymphocyte differentiation, activation, and effector function. Increases in cytosolic-free Ca(2+) concentrations are thought to be mediated through two interconnected and complementary mechanisms: the release of endoplasmic reticulum Ca(2+) "stores" and "store-operated" Ca(2+) entry via plasma membrane channels. However, the identity of molecular components conducting Ca(2+) currents within developing and mature T?cells is unclear. Here, we have demonstrated that the L-type "voltage-dependent" Ca(2+) channel Ca(V)1.4 plays a cell-intrinsic role in the function, development, and survival of naive T?cells. Plasma membrane Ca(V)1.4 was found to be essential for modulation of intracellular Ca(2+) stores and T?cell receptor (TCR)-induced rises in cytosolic-free Ca(2+), impacting activation of Ras-extracellular signal-regulated kinase (ERK) and nuclear factor of activated T?cells (NFAT) pathways. Collectively, these studies revealed that Ca(V)1.4 functions in controlling naive T?cell homeostasis and antigen-driven T?cell immune responses.     

The functional network of ion channels in T lymphocytes

Summary:  For more than 25 years, it has been widely appreciated that Ca²⁺ influx is essential to trigger T-lymphocyte activation. Patch clamp analysis, molecular identification, and functional studies using blockers and genetic manipulation have shown that a unique contingent of ion channels orchestrates the initiation, intensity, and duration of the Ca²⁺ signal. Five distinct types of ion channels – Kv1.3, KCa3.1, Orai1+ stromal interacting molecule 1 (STIM1) [Ca²⁺-release activating Ca²⁺ (CRAC) channel], TRPM7, and Cl_swell– comprise a network that performs functions vital for ongoing cellular homeostasis and for T-cell activation, offering potential targets for immunomodulation. Most recently, the roles of STIM1 and Orai1 have been revealed in triggering and forming the CRAC channel following T-cell receptor engagement. Kv1.3, KCa3.1, STIM1, and Orai1 have been found to cluster at the immunological synapse following contact with an antigen-presenting cell; we discuss how channels at the synapse might function to modulate local signaling. Immuno-imaging approaches are beginning to shed light on ion channel function in vivo . Importantly, the expression pattern of Ca²⁺ and K⁺ channels and hence the functional network can adapt depending upon the state of differentiation and activation, and this allows for different stages of an immune response to be targeted specifically.     

Pharmacological and biophysical properties of Ca2+ channels and subtype distributions in human adrenal chromaffin cells

In this study, we explored the pharmacological and biophysical properties of voltage-activated Ca(2+) channels in human chromaffin cells using the perforated-patch configuration of the patch-clamp technique. According to their pharmacological sensitivity to Ca(2+) channel blockers, cells could be sorted into two groups of similar size showing the predominance of either N- or P/Q-type Ca(2+) channels. R-type Ca(2+) channels, blocked by 77% with 20 muM Cd(2+) and not affected by 50 muM Ni(2+), were detected for the first time in human chromaffin cells. Immunocytochemical experiments revealed an even distribution of alpha (1E) Ca(2+) channels in these cells. With regard to their biophysical properties, L- and R-type channels were activated at membrane potentials that were 15-20 mV more negative than P/Q- and N-type channels. Activation time constants showed no variation with voltage for the L-type channels, decreased with increasing potentials for the R- and P/Q-type channels, and displayed a bell shape with a maximum at 0 mV for the N-type channels. R-type channels were also the most inactivated channels. We thus show here that human chromaffin cells possess all the Ca(2+) channel types described in neurons, L, N, P/Q, and R channels, but the relative contributions of N and P/Q channels differ among cells. Given that N- and P/Q-type Ca(2+) channel types can be differentially modulated, these findings suggest the possibility of cell-specific regulation in human chromaffin cells.     

Ion channel gene expression in human lung,skin, and cord blood-derived mast cells

Immunoglobulin E (IgE)-dependent activation of human mast cells (HMC) is characterized by an influx of extracellular calcium (Ca(2+)), which is essential for subsequent release of preformed (granule-derived) mediators and newly generated autacoids and cytokines. In addition, flow of ions such as K(+) and Cl(-) is likely to play an important role in mast cell activation, proliferation, and chemotaxis through their effect on membrane potential and thus Ca(2+) influx. It is therefore important to identify these critical molecular effectors of HMC function. In this study, we have used high-density oligonucleotide probe arrays to characterize for the first time the profile of ion channel gene expression in human lung, skin, and cord blood-derived mast cells. These cells express mRNA for inwardly rectifying and Ca(2+)-activated K(+) channels, voltage-dependent Na(+) and Ca(2+) channels, purinergic P2X channels, transient receptor potential channels, and voltage-dependent and intracellular Cl(-) channels. IgE-dependent activation had little effect on ion channel expression, but distinct differences for some channels were observed between the different mast cell phenotypes, which may contribute to the mechanism of functional mast cell heterogeneity.     

Ionic factors governing rebound burst phenotype in rat deep cerebellar neurons

Large diameter cells in rat deep cerebellar nuclei (DCN) can be distinguished according to the generation of a transient or weak rebound burst and the expression of T-type Ca(2+) channel isoforms. We studied the ionic basis for the distinction in burst phenotypes in rat DCN cells in vitro. Following a hyperpolarization, transient burst cells generated a high-frequency spike burst of < or = 450 Hz, whereas weak burst cells generated a lower-frequency increase (<140 Hz). Both cell types expressed a low voltage-activated (LVA) Ca(2+) current near threshold for rebound burst discharge (-50 mV) that was consistent with T-type Ca(2+) current, but on average 7 times more current was recorded in transient burst cells. The number and frequency of spikes in rebound bursts was tightly correlated with the peak Ca(2+) current at -50 mV, showing a direct relationship between the availability of LVA Ca(2+) current and spike output. Transient burst cells exhibited a larger spike depolarizing afterpotential that was insensitive to blockers of voltage-gated Na(+) or Ca(2+) channels. In comparison, weak burst cells exhibited larger afterhyperpolarizations (AHPs) that reduced cell excitability and rebound spike output. The sensitivity of AHPs to Ca(2+) channel blockers suggests that both LVA and high voltage-activated (HVA) Ca(2+) channels trigger AHPs in weak burst compared with only HVA Ca(2+) channels in transient burst cells. The two burst phenotypes in rat DCN cells thus derive in part from a difference in the availability of LVA Ca(2+) current following a hyperpolarization and a differential activation of AHPs that establish distinct levels of membrane excitability.     

The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry

Stimulation of membrane receptors linked to a phospholipase C and the subsequent production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (InsP(3)) is a signaling pathway of fundamental importance in eukaryotic cells. Signaling downstream of these initial steps involves mobilization of Ca(2+) from intracellular stores and Ca(2+) influx through the plasma membrane. For this influx, several contrasting mechanisms may be responsible but particular relevance is attributed to the induction of Ca(2+) influx as consequence of depletion of intracellular calcium stores. This phenomenon (frequently named store-operated calcium entry, SOCE), in turn, may be brought about by various signals, including soluble cytosolic factors, interaction of proteins of the endoplasmic reticulum with ion channels in the plasma membrane, and a secretion-like coupling involving translocation of channels to the plasma membrane. Experimental approaches to analyze these mechanisms have been considerably advanced by the discovery of mammalian homologs of the Drosophila cation channel transient receptor potential (TRP). Some members of the TRP family can be expressed to Ca(2+)-permeable channels that enable SOCE; other members form channels activated independently of stores. TRP proteins may be an essential part of endogenous Ca(2+) entry channels but so far expression of most TRP cDNAs has not resulted in restitution of channels found in any mammalian cells, suggesting the requirement for further unknown subunits. A major exception is CaT1, a TRP channel demonstrated to provide Ca(2+)-selective, store-operated currents identical to those characterized in several cell types. Ongoing and future research on TRP channels will be crucial to understand the molecular basis of receptor-mediated Ca(2+) entry, with respect to the structure of the entry channels as well as to the mechanisms of its activation and regulation.     

Chemical anoxia activates ATP-sensitive and blocks Ca(2+)-dependent K(+) channels in rat dorsal vagal neurons in situ

The contribution of subclasses of K(+) channels to the response of mammalian neurons to anoxia is not yet clear. We investigated the role of ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) currents (small conductance, SK, big conductance, BK) in mediating the effects of chemical anoxia by cyanide, as determined by electrophysiological analysis and fluorometric Ca(2+) measurements in dorsal vagal neurons of rat brainstem slices. The cyanide-evoked persistent outward current was abolished by the K(ATP) channel blocker tolbutamide, but not changed by the SK and BK channel blockers apamin or tetraethylammonium. The K(+) channel blockers also revealed that ongoing activation of K(ATP) and SK channels counteracts a tonic, spike-related rise in intracellular Ca(2+) ([Ca(2+)](i)) under normoxic conditions, but did not modify the rise of [Ca(2+)](i) associated with the cyanide-induced outward current. Cyanide depressed the SK channel-mediated afterhyperpolarizing current without changing the depolarization-induced [Ca(2+)](i) transient, but did not affect spike duration that is determined by BK channels. The afterhyperpolarizing current and the concomitant [Ca(2+)](i) rise were abolished by Ca(2+)-free superfusate that changed neither the cyanide-induced outward current nor the associated [Ca(2+)](i) increase. Intracellular BAPTA for Ca(2+) chelation blocked the afterhyperpolarizing current and the accompanying [Ca(2+)](i) increase, but had no effect on the cyanide-induced outward current although the associated [Ca(2+)](i) increase was noticeably attenuated. Reproducing the cyanide-evoked [Ca(2+)](i) transient with the Ca(2+) pump blocker cyclopiazonic acid did not evoke an outward current.Our results show that anoxia mediates a persistent hyperpolarization due to activation of K(ATP) channels, blocks SK channels and has no effect on BK channels, and that the anoxic rise of [Ca(2+)](i) does not interfere with the activity of these K(+) channels.     

Activation of large-conductance Ca(2+)-activated K(+) channels depresses basal synaptic transmission in the hippocampal CA1 area in APP (swe/ind) TgCRND8 mice

Large-conductance Ca(2+)-activated K(+) (BK) channels regulate synaptic transmission by contributing to the repolarization phase of the action potential that invades the presynaptic terminal. BK channels are prone to activation under pathological conditions, such as brain ischemia and epilepsy. It is unclear if activation of these channels contributes to the depression of synaptic transmission observed in the early stage of Alzheimer's disease (AD). In this study, we recorded the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus CA1 region of brain slices from 6 to 9 weeks (pre-plaque) TgCRND8 mice, a mouse model of Alzheimer's disease that harbors a double amyloid precursor mutation (KM670N/671L "Swedish" and V717F "Indiana"). Compared to age-matched controls, the fEPSPs in these animals are significantly depressed. This depression is largely mediated by the activation of presynaptic BK channels in the CA1 area. Both BK channel blockers (charybdotoxin and paxilline), and the fast binding calcium chelator, BAPTA-AM, enhance the fEPSP by deactivating the BK channels. Repetitive stimulation to the afferent pathway enhances fEPSP. This enhancement is more prominent when BK channel blockers are added in Tg slices, suggesting that repetitive stimulation further promotes BK channel activation in Tg slices. The potential candidates that mediate the activation of BK channels in these pre-plaque Alzheimer's disease model mice might involve impaired calcium homeostasis and AD related over-generation of reactive oxygen species.     

Characterization of Ca(2+) channels in rat subthalamic nucleus neurons

The subthalamic nucleus (STN) plays a key role in motor control. Although previous studies have suggested that Ca(2+) conductances may be involved in regulating the activity of STN neurons, Ca(2+) channels in this region have not yet been characterized. We have therefore investigated the subtypes and functional characteristics of Ca(2+) conductances in STN neurons, in both acutely isolated and slice preparations. Acutely isolated STN cells were identified by retrograde filling with the fluorescent dye, Fluoro-Gold. In acutely isolated STN neurons, Cd(2+)-sensitive, depolarization-activated Ba(2+) currents were observed in all cells studied. The current-voltage relationship and current kinetics were characteristic of high-voltage-activated Ca(2+) channels. The steady-state voltage-dependent activation curves and inactivation curves could both be fitted with a single Boltzmann function. Currents evoked with a prolonged pulse, however, inactivated with multiple time constants, suggesting either the presence of more than one Ca(2+) channel subtype or multiple inactivation processes with a single channel type in STN neurons. Experiments using organic Ca(2+) channel blockers revealed that on average, 21% of the current was nifedipine sensitive, 52% was sensitive to omega-conotoxin GVIA, 16% was blocked by a high concentration of omega-agatoxin IVA (200 nM), and the remainder of the current (9%) was resistant to the co-application of all blockers. These currents had similar voltage dependencies, but the nifedipine-sensitive current and the resistant current activated at slightly lower voltages. omega-Agatoxin IVA at 20 nM was ineffective in blocking the current. Together, the above results suggest that acutely isolated STN neurons have all subtypes of high-voltage-activated Ca(2+) channels except for P-type, but have no low-voltage-activated channels. Although acutely isolated neurons provide a good preparation for whole cell voltage-clamp study, dendritic processes are lost during dissociation. To gain information on Ca(2+) channels in dendrites, we thus studied Ca(2+) channels of STN neurons in a slice preparation, focusing on low-voltage-activated channels. In current-clamp recordings, a slow spike was always observed following termination of an injected hyperpolarizing current. The slow spike occurred at resting membrane potentials and was sensitive to micromolar concentrations of Ni(2+), suggesting that it is a low-threshold Ca(2+) spike. Together, our results suggest that STN neurons express low-voltage-activated Ca(2+) channels and several high-voltage-activated subtypes. Our results also suggest the possibility that the low-voltage-activated channels have a preferential distribution to the dendritic processes.     

T-type calcium channel blockers that attenuate thalamic burst firing and suppress absence seizures

Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca(2+)) channels. We sought to develop high-affinity T-type specific Ca(2+) channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca(2+) channel pharmacophore and a high-throughput fluorometric Ca(2+) influx assay, we identified the T-type Ca(2+) channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca(2+) channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.