Effects of cholesterol levels on the excitability of rat hippocampal neurons

Changes in the cholesterol levels dynamically alter the microenvironment of the plasma membrane and have been shown to modify functions of ion channels. However, the cellular effect of these modifications is largely unknown. In this report, we demonstrate that cholesterol levels modulate neuronal excitability in rat hippocampal neurons. Reduction of cholesterol levels shortened the duration and increased the firing frequency and peak amplitude of action potentials, while enrichment of cholesterol reversed the effect. Furthermore, we showed that reduction of cholesterol levels increased, while enrichment of cholesterol decreased the amplitude of the delayed rectifier I_K currents. On the other hand, reduction of cholesterol levels slowed down the inactivation of the fast transient I_A currents, but enrichment of cholesterol had no significant effect on the I_A currents. Besides, alteration in cholesterol levels had no significant effect on the action potential in the presence of blockers for both I_K and I_A currents. These observations demonstrate that cholesterol levels bi-directionally regulate the neuronal excitability mainly through modifications of the I_K and I_A currents, suggesting an optimum level of cholesterol for the optimum excitability of neurons. Alterations in the neuronal cholesterol levels have been associated with aging, cognitive decline, neurodegenerative diseases, etc. Therefore, our findings are important for a deeper understanding of the relationship between the cholesterol level and dysfunctions of the brain at the molecular level.     

Role of an A-Type K+ Conductance in the Back-Propagation of Action Potentials in the Dendrites of Hippocampal Pyramidal Neurons

Action potentials elicited in the axon actively back-propagate into the dendritic tree. During this process their amplitudes can be modulated by internal and external factors. We used a compartmental model of a hippocampal CA1 pyramidal neuron to illustrate how this modulation could depend on (1) the properties of an A-type K⁺ conductance that is expressed at high density in hippocampal dendrites and (2) the relative timing of synaptic activation. The simulations suggest that the time relationship between pre- and postsynaptic activity could help regulate the amplitude of back-propagating action potentials, especially in the distal portion of the dendritic tree.     

无血清原代培养大鼠海马神经元的形态与膜电位

分离新生Wistar鼠海马,采用添加B27的无血清培养液进行海马神经元原代培养,动态观察海马神经元形态学变化;通过免疫荧光细胞化学法检测神经纤丝(NF)的表达,进行神经元鉴定及纯度计算;采用电位敏感的荧光探针标记神经元,在激光扫描共聚焦显微镜上动态监测去极化剂KCl作用前后膜电位的变化,观察神经元电生理反应。结果表明:此方法培养的大鼠海马神经元可在体外存活20天以上,9～14天为发育最成熟阶段,培养7天神经元纯度达90%。KCl作用于细胞后胞内荧光强度增强,细胞迅速去极化。本培养方法在体外获得高纯度的海马神经元并延长体外存活时间,且显示出神经元的电生理反应特性。     

乌拉坦对大鼠海马CA1神经元电压门控钠通道和动作电位的作用

乌拉坦对兴奋性和抑制性配体门控通道具有广泛的可检测的作用.作者运用全细胞膜片钳技术研究乌拉坦对wistar大鼠海马CA1神经元电压门控钠通道和动作电位的作用.结果发现乌拉坦可逆并剂量依赖性地抑制钠电流和动作电位,其中,在10mmol/L浓度时可减小钠电流强度达38%,使激活曲线向去极化方向移动,并延长钠通道失活后的恢复时间,降低动作电位的幅值.这些结果表明乌拉坦对电压门控钠通道的抑制作用可能是乌拉坦全身麻醉作用的机制之一.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

短暂脑缺血大鼠海马CA1区锥体细胞大电导Ca2+依赖K+通道活动降低(英)

短暂脑缺血可对随后的损伤性脑缺血表现出明显的耐受.有研究表明大电导Ca²⁺依赖K⁺（BK_Ca）通道活动增强参与了缺血性脑损伤.采用膜片钳的内面向外式,观察了3 min短暂脑缺血后6 h、24 h以及48 h大鼠海马CA1区锥体细胞上BK_Ca通道活动的动态变化.短暂脑缺血后BK_Ca通道的单通道电导和翻转电位均未见明显变化,但通道的开放概率则在缺血预处理后的前24 h内显著降低.通道动力学分析显示通道关闭时间变长是短暂脑缺血后通道活动降低的主要原因,因为通道的开放时间未发生明显变化.结果提示短暂脑缺血所致的BK_Ca通道活动降低可能与缺血耐受的产生有关.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Membrane channels as integrators of G-protein-mediated signaling

A variety of extracellular stimuli regulate cellular responses via membrane receptors. A well-known group of seven-transmembrane domain-containing proteins referred to as G protein-coupled receptors, directly couple with the intracellular GTP-binding proteins (G proteins) across cell membranes and trigger various cellular responses by regulating the activity of several enzymes as well as ion channels. Many specific populations of ion channels are directly controlled by G proteins; however, indirect modulation of some channels by G protein-dependent phosphorylation events and lipid metabolism is also observed. G protein-mediated diverse modifications affect the ion channel activities and spatio-temporally regulate membrane potentials as well as of intracellular Ca^2 + concentrations in both excitatory and non-excitatory cells. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Functional role of the activity of ATP-sensitive potassium channels in electrical behavior of hippocampal neurons: experimental and theoretical studies

ATP-sensitive K⁺ (K_ATP) channels are distributed in a variety of cell types, including hippocampal neurons. These channels provide a link between electrical activity of cell membranes and cellular metabolism. The activity of K_ATP channels in hippocampal H19-7 neurons treated with or without short interfering RNAs (siRNAs) directed against Kir6.2 mRNA was investigated in this study. In single-channel recordings, cell exposure to diazoxide (30 μM) significantly prolonged the mean open time of K_ATP channels; however, neither closed-time kinetics nor the single-channel conductance of the channel was altered by this compound. However, in cells transfected with Kir6.2 siRNAs, diazoxide-stimulated activity of K_ATP channels was abolished. Based on single-channel recordings, the activity of K_ATP channels was mathematically constructed in a Markovian manner. The simulated activity of single K_ATP channels was incorporated in a modeled hippocampal neuron to assess how any changes in K_ATP-channel activity affect burst firing of action potentials (APs). The modeled neuron was adopted from the model of Xu and Clancy (2008). Specifically, to mimic the action of diazoxide, the baseline value of open time (τ_bas) of K_ATP channels was arbitrarily elevated, while varying number of active channels (N_O) was set to simulate electrical behavior of Kir6.2 siRNAs-transfected cells. The increase of either N_O or τ_bas depressed membrane excitability of modeled neuron. Fast-slow analysis of AP bursting from this modeled neuron also revealed that the increased K_ATP-channel activity shifted the voltage nullcline in an upward direction, thereby leading to a reduction of the repetitive spike regime. Taken together, it is anticipated that the increased activity of K_ATP channels caused by increasing N_O or τ_bas contributes to or is responsible for burst firing of APs in hippocampal neurons if similar results occur in vivo.     

Human ether-a-go-go related gene (hERG) K channels: Function and dysfunction

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in regulating cardiac excitability and maintenance of normal cardiac rhythm. Mutations in hERG cause a third of all cases of congenital long QT syndrome, a disorder of cardiac repolarisation characterised by prolongation of the QT interval on the surface electrocardiogram, abnormal T waves, and a risk of sudden cardiac death due to ventricular arrhythmias. Additionally, the hERG channel protein is the molecular target for almost all drugs that cause the acquired form of long QT syndrome. Advances in understanding the structural basis of hERG gating, its traffic to the cell surface, and the molecular architecture involved in drug-block of hERG, are providing the foundation for rational treatment and prevention of hERG associated long QT syndrome. This review summarises the current knowledge of hERG function and dysfunction, and the areas of ongoing research.     

Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell current recording

Summary The effects of tetraethylammonium ions on currents through high-conductance voltage- and Ca²⁺-activated K⁺ channels have been studied with the help of patch-clamp single-channel and whole-cell current recording on pig pancreatic acinar cells. In excised outside-out membrane patches TEA (1 to 2 mM) added to the bath solution virtually abolishes unitary current activity except at very positive membrane potentials when unitary currents corresponding to a markedly reduced conductance are observed. TEA in a lower concentration (0.2 mM) markedly reduces the open-state probability and causes some reduction of the single-channel conductance. In inside-out membrane patches bath application of TEA in concentrations up to 2 mM has no effect on single-channel currents. At a higher concentration (10 mM) slight reductions in single-channel conductance occur. In whole-cell current recording experiments TEA (1 to 2 mM) added to the bath solution completely suppresses the outward currents associated with depolarizing voltage jumps to membrane potentials of 0 mV and blocks the major part (70 to 90%) of the outward currents even at very positive membrane potentials (30 to 40 mV). In contrast TEA (2 mM) added to the cell interior (pipette solution) has no effect on the outward K⁺ current. Our results demonstrate that TEA in low concentrations (1 to 2 mM) acts specifically on the outside of the plasma membrane to block current through the high-conductance Ca²⁺- and voltage-activated K⁺ channels     

Nitric Oxide Links the Apical Na+ Transport to the Basolateral K+ Conductance in the Rat Cortical Collecting Duct

We have used the patch clamp technique to study the effects of inhibiting the apical Na⁺ transport on the basolateral small-conductance K⁺ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nP _o (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na⁺ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na⁺ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca²⁺ because removal of Ca²⁺ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca²⁺, we observed that amiloride and benzamil significantly decreased intracellular Ca²⁺ in the Ca²⁺-containing solution but had no effect in a Ca²⁺-free bath. Furthermore, raising intracellular Ca²⁺ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca²⁺ are responsible for the effects on SK activity of inhibition of the Na⁺ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca²⁺ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca²⁺-free bath solution. We conclude that Ca²⁺-dependent NO generation mediates the effect of inhibiting the apical Na⁺ transport on the basolateral SK in the rat CCD.     

Axonal excitability revisited

The original papers of Hodgkin and Huxley (J. Physiol. 116 (1952a) 449, J. Physiol. 116 (1952b) 473, J. Physiol. 116 (1952c) 497, J. Physiol. 117 (1952d) 500) have provided a benchmark in our understanding of cellular excitability. Not surprisingly, their model of the membrane action potential (AP) requires revisions even for the squid giant axon, the preparation for which it was originally formulated. The mechanisms they proposed for the voltage-gated potassium and sodium ion currents, IK, and INa, respectively, have been superceded by more recent formulations that more accurately describe voltage-clamp measurements of these components. Moreover, the current-voltage relation for IK has a non-linear dependence upon driving force that is well described by the Goldman-Hodgkin-Katz (GHK) relation, rather than the linear dependence on driving force found by Hodgkin and Huxley. Furthermore, accumulation of potassium ions in the extracellular space adjacent to the axolemma appears to be significant even during a single AP. This paper describes the influence of these various modifications in their model on the mathematically reconstructed AP. The GHK and K+ accumulation results alter the shape of the AP, whereas the modifications in IK and INa gating have surprisingly little effect. Perhaps the most significant change in their model concerns the amplitude of INa, which they appear to have overestimated by a factor of two. This modification together with the GHK and the K+ accumulation results largely remove the discrepancies between membrane excitability of the squid giant axon and the Hodgkin and Huxley (J. Physiol. 117 (1952d) 500) model previously described (Clay, J. Neurophysiol. 80 (1998) 903).     

Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1

The K⁺ channel Kcv is encoded by the chlorella virus PBCV-1. There is evidence that this channel plays an essential role in the replication of the virus, because both PBCV-1 plaque formation and Kcv channel activity in Xenopus oocytes have similar sensitivities to inhibitors. Here we report circumstantial evidence that the Kcv channel is important during virus infection. Recordings of membrane voltage in the host cells Chlorella NC64A reveal a membrane depolarization within the first few minutes of infection. This depolarization displays the same sensitivity to cations as Kcv conductance; depolarization also requires the intact membrane of the virion. Together these data are consistent with the idea that the virus carries functional K⁺ channels in the virion and inserts them into the host cell plasma membrane during infection.     

Characterization of Hydrogen Peroxide Toxicity in Cultured Rat Forebrain Neurons

We investigated the ability of hydrogen peroxide (H₂O₂) to cause apoptotic cell death in cultured rat forebrain neurons and the potential mechanisms by which oxidative stress triggers delayed neuronal death. H₂O₂ (25 M for 5 min) reduced cell viability to 34.5 ± 8.3% of untreated controls 20 h after exposure, and resulted in a significant proportion of neurons which exhibited apoptotic nuclear morphology. Using single cell fluorescence assays, we measured H₂O₂-induced changes in DNA strand breaks, 27 dichlorofluorescin fluorescence, reduced glutathione, intracellular free Ca²⁺, and mitochondrial membrane potential. DNA strand breaks in response to H₂O₂ were not evident immediately following exposure, but were increased 12h and 20h after exposure. Millimolar concentrations of H₂O₂ caused increases in the fluorescence of the oxidant-sensitive fluorescent dye, 27-dichlorofluorescin. H₂O₂ treatment decreased reduced glutathione following 30 minutes of exposure using the fluorescent indicator, 5-chloromethylfluorescein diacetate, and increased intra-neuronal free Ca²⁺ levels in a subpopulation of neurons. Mitochondrial membrane potential, measured by rhodamine 123 localization was unaffected by 25 H₂O₂, while higher concentrations of H₂O₂ (10 or 30 mM) depolarized mitochondria. These studies demonstrate that H₂O₂ is a potent and effective neurotoxin that produces oxidative stress, as well as apoptotic neuronal death     

Ca<Superscript>2+</Superscript> Buffering Capacity of Mitochondria After Oxygen-Glucose Deprivation in Hippocampal Neurons

In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca²⁺ ([Ca²⁺]_c) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca²⁺influx. The [Ca²⁺]_c level increased sharply, followed by a rapid increase in mitochondrial Ca²⁺ [Ca²⁺]_m. The increase in the [Ca²⁺]_m level accompanied a reduction in the [Ca²⁺]_c level. After reaching a peak, the [Ca²⁺]_c level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.     

Potential role of potassium channel openers in the treatment of asthma and chronic obstructive pulmonary disease

Airway smooth muscle (ASM) cells express various types of potassium (K+) channels which play a key role in determining the resting membrane potential, a relative electrical stability and the responsiveness to both contractile and relaxant agents. In addition, K+ channels are also involved in modulation of neurotransmitter release from airway nerves. The most important K+ channels identified in airways include large and small Ca2+-activated, delayed-rectifier, and ATP-sensitive channels. These K+ channels are structurally and functionally different, thus playing distinct roles in airway electrophysiology and pharmacology. Many in vitro and in vivo studies, performed in both animals and humans, have shown that K+ channel openers are able to induce hyperpolarization of ASM cells, bronchodilation, suppression of airway hyperresponsiveness (AHR), and inhibition of neural reflexes. Therefore, airway K+ channels represent a suitable pharmacological target for the development of new effective therapeutic options in the treatment of asthma and chronic obstructive pulmonary disease (COPD).     

Evidence for a K+ channel in bovine chromaffin granule membranes: single-channel properties and possible bioenergetic significance

A K⁺ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (ghosts). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of 400 pS in symmetric 450 mM KCI, with the permeability sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, and was insensitive to both Ca²⁺ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA⁺ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA⁺-sensitive K⁺ channel which may have a role in regulating the vesicle membrane potential. Correspondence to: R. H. Ashley     

Vernakalant activates human cardiac K2P17.1 background K channels

Atrial fibrillation (AF) contributes significantly to cardiovascular morbidity and mortality. The growing epidemic is associated with cardiac repolarization abnormalities and requires the development of more effective antiarrhythmic strategies. Two-pore-domain K⁺ channels stabilize the resting membrane potential and repolarize action potentials. Recently discovered K_2P17.1 channels are expressed in human atrium and represent potential targets for AF therapy. However, cardiac electropharmacology of K_2P17.1 channels remains to be investigated. This study was designed to elucidate human K_2P17.1 regulation by antiarrhythmic drugs.