Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

细胞骨架与血糖调节

细胞骨架由微丝、微管和中间丝构成,参与血糖调节这一复杂的生理过程,在胰岛素分泌、胰岛素功能和糖代谢相关酶类的细胞内分布等方面具有重要的作用。本文将从以上三个方面,对细胞骨架与血糖调节的关系加以综述。     

Ion channels in murine nuclei during early development and in fully differentiated adult cells

Summary The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores.     

新霉素抑制PC12细胞烟碱受体介导的反应

新霉素是一种氨基甙类抗生素,在细胞水平可以抑制磷脂酶C介质的信号转导系统,本研究采用全细胞膜片钳技术,以大鼠肾上腺嗜铬细胞瘤细胞（PC12)为标本,观察了新霉素参考书国酰胆碱诱发电流（IACh)的影响,药理学鉴定表明,PC12细胞上的IACh是通过ACh激动烟碱受体引起的,钳制电压为－80mV时,ACh(30umol/L)诱发一内向电流;细胞外同时给予新霉素（0．01－1mmol/L)和ACh(30μmol/L)可显著抑制IACh峰值,此抑制作用迅速,可逆,呈浓度依赖性,用新霉素预处理细胞3－8min不影响其对IACh的抑制作用,用外源性蛋白激酶C（PKC）激剂激活PKC,同样可抑制IACh,而细胞内透析PKC抑制剂（PKCI19-31,0.1-5μmol/L）不影响新霉素对IACh的抑制作用,以上结果提示,新霉对PC12细胞的IACh的有抑制作用,这是一种与磷脂酶C阻断无关的药理学效应。     

Regulation of the microfilament system in normal and polyoma virus transformed cultured (BHK) cells

The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30-50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and alpha-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 degrees C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of electrophysiologically distinct subpopulations of rat taste cells

Summary The gustatory sensory system provides animals with a rapid chemical analysis of a potential food substance providing information necessary to facilitate ingestion or rejection of the food. The process of gustatory transduction is initiated in the taste cells in the lingual epithelium. However, due to the small size, scarcity of the cells and their location, embedded in a keratinized squamous epithelium, it has been difficult to study the primary events in the transduction process. Recently, we have developed a preparation of dissociated rat taste cells that permits studies of the taste transduction process in single isolated cells. We have now investigated the electrophysiological properties of the rat taste cells using the patch-clamp technique. We have identified two populations of cells within the taste bud: one expressing a voltage-dependent potassium current and the second containing both voltage-dependent sodium and potassium currents. The potassium current in both cell groups is blocked by external TEA, Ba²⁺, and quinine. Two types of K⁺ channels have been identified: a 90-pS delayed rectifier K⁺ channel and a maxi calcium-activated K⁺ channel. The sodium current is blocked by TTX, but not by amiloride.     

神经生长因子分化后的PC12 细胞对乙酰胆碱的敏感性及其特性

目的和方法：采用全细胞膜片钳技术观察神经生长因子（NGF）分化后的PC12细胞对乙酰胆碱（ACh)的敏感性,并对ACh诱发电流（IACh)的特性进行分析。结果：NGF处理后的PC12乐仅形态上向交感神经元分化,而且具有电学兴奋性,它对ACh敏感性比未分化前显著提高。药理学鉴定表明PC12上的IACh是由烟碱受体（nAChR)引起的,具有明显的失敏特性。宏观IACh呈内向整流和浓度依赖性。结论：PC12细胞培养方便,同源性好,加入NGF后向交感神经元分化,且其具有神经元烟碱受体,可以作为交感神经元烟碱受体研究的很好的模型系统。     

Differential regulation of K+ channels in Arabidopsis epidermal and stelar root cells

The patch clamp technique was applied to protoplasts isolated from the epidermis and pericycle of Arabidopsis roots and their plasma membrane currents investigated. In the whole cell configuration, all protoplasts from the epidermis exhibited depolarization‐activated time‐dependent outwardly rectifying (OR) currents whereas OR currents were present in only 50% of cells from the pericycle. The properties of the OR currents in the epidermis and pericycle were compared with respect to their selectivity, pharmacology and gating. The time‐dependent activation kinetics, selectivity and sensitivity to extracellular tetraethyl ammonium of the OR current in each cell type were not significantly different. The reversal potential (E_rev) of the OR currents indicated that they were primarily due to the movement of K⁺. However, the gating properties of the OR currents from the epidermis differed markedly from those exhibited in the pericycle. Although both cell types displayed OR currents with voltage‐dependent gating modulated in a potassium‐dependent fashion [i.e. the activation threshold (V_0.5) was displaced to more positive voltages as extracellular K⁺ increased], the OR currents in the epidermis also displayed voltage‐independent gating by extracellular K⁺ which dramatically regulated current density. In the present study, reducing extracellular K⁺ activity from 40 to 0.87 mm reduced the OR current density in epidermal cells by approximately 80%. The chord conductance of the OR current saturated as a function of extracellular K⁺ and could be fitted with a Michaelis–Menten function to yield a binding constant (K_m) of 10.5 mm . The ability of other monovalent cations to substitute for K⁺‐gating of the OR currents was also investigated and shown to exhibit a relative sequence of K⁺ ≥ Rb⁺ > Cs⁺ > Na⁺ ≥ Li⁺ (Eisenmann sequence IV) with respect to efficacy of gating. Furthermore, single channel recordings demonstrated that channel activity rather than the single channel conductance was modulated by extracellular K⁺. In contrast, OR current density in the pericycle was largely independent of extracellular K⁺. It is suggested that the contrasting gating properties of the K⁺ channels in the epidermis and pericycle reflect their different physiological roles, particularly with respect to their role in K⁺ (nutrient) transport from the soil solution to the shoot.     

Properties of single voltage-gated proton channels in human eosinophils estimated by noise analysis and by direct measurement

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi < or = 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, sigmaH2. We analyze sigmaH2 data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic gH was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi < or = 5.5 near Vthreshold (the voltage at which gH turns on), single channel-like current events were observed with amplitudes 7-16 fA.     

Chloride channels on epithelial cells cultured from human fetal epididymis

Summary Using single-channel recording techniques, we have detected two types of outwardly rectifying chloride channel on epithelial cells cultured from human fetal epididymis. A small-conductance channel (2.8–5.0 pS) was spontaneously active in 29% of cell-attached patches but rapidly disappeared on patch excision. This channel often occurred in clusters and exhibited slow kinetics with open and closed times of the order of tens or hundreds of msec; an open-state probability that was essentially independent of voltage; and a very low permeability to bicarbonate relative to chloride. Exposing epididymal cells to either forskolin (3 m) or adrenaline (1 m) activated this channel (up to 350-fold), suggesting that it may be involved in cyclic AMP-mediated anion secretion by the male reproductive tract. The large-conductance channel (14 to 29 pS) was never detected in cell-attached patches but could be activated by depolarization (40 mV) in 3% of excised, inside-out patches. Once activated, opening of this large channel was voltage independent, and it had a relatively high permeability to both gluconate (P _gluconate/P _chloride=0.24) and bicarbonate (P _bicarbonate/P _chloride=0.4). The proportion of excised patches that contained this channel was increased 2.5-fold by prior stimulation of the epididymal cells; however, because the channel was never observed in cell-attached patches its physiological role must remain uncertain.     

Intracellular activities during volume regulation byNecturus gallbladder

Summary Necturus gallbladder epithelial cells regulate their volume after a change in solution osmolality. We determined the intracellular activities of Na, K and Cl when the mucosal bathing solution osmolality was increased 18% by the addition of mannitol. The gallbladder was mounted in a rapid flow chamber and punctured simultaneously with two single-barrelled microelectrodes. One electrode sensed membrane potential and the other was sensitive to the activity of Na, K or Cl. Cell volume measurements, made in previous studies utilizing quantitative light microscopy, indicated that hypertonicity of the mucosal bath first caused a cell shrinkage of 15% followed by volume readjustment. Some loss of Na, K and Cl was observed during shrinkage; subsequently during volume regulation, the intracellular quantities of all three ions increased. The loss of Na during the initial cell shrinkage could be blocked by ouabain and was therefore due to increased transport. K and Cl losses were probably related to the increase in their concentrations during shrinkage. The gain of Na, K and Cl during volume regulation was similar in magnitude to the loss of these solutes during cell shrinkage. The increase of Na, K and Cl during volume regulation accounted for about 60% of the increase of cell solutes during this period indicating that other solutes also contributed to the volume regulation response.     

Chloride channels in the nuclear membrane

Summary Chloride-selective ion channels were measured from isolated rat liver nuclei. Single ion channel currents were recorded in both nuclear-attached and in excised patches in the insideout configuration of the patch-clamp technique. Two types of chloride conductance were defined, a large conductance (150 pS;i _Cl.N ) channel with complex kinetics and multiple substates, and a second smaller conductance (58 pS;I _Cl.n ) channel sensitive to block by ATP. The channels were inhibited by pharmacological agents known to block chloride channels and were insensitive to internal and external changes in calcium and magnesium. Presumably the channels reside in the external membrane of the nuclear double membrane and may mediate charge balance in the release and uptake of calcium from the perinuclear space.     

ATP激活鼻咽癌细胞氯电流并减小细胞容积

采用全细胞膜片钳技术和细胞容积测量技术,在低分化鼻咽癌细胞株CNE-2Z上观察ATP 诱导的Cl- 电流的特性及其对细胞容积的影响。细胞外微摩尔水平的ATP 以剂量依赖性的方式激活一个具有弱外向整流特性,没有时间依赖性失活的电流,此电流的反转电位 [(-0.05 ± 0.03) mV]接近Cl- 的平衡电位(-0.9 mV)。用葡萄糖酸置换细胞外液Cl- 后, ATP 激活的电流明显减小并且反转电位发生改变。氯通道抑制剂NPPB (200 μmol/L)可以抑制这一电流 [(81.03 ± 9.3)%] 。此电流亦可被嘌呤受体(P2Y) 拮抗剂反应蓝 2 抑制 [(67.39 ± 5.06)%]。50 μmol/L 的 ATP 使在等渗状态下的细胞容积缩小, 替代和耗竭细胞外、内的Cl- 后, ATP 的这一作用消失。这些结果提示细胞外微摩尔水平的 ATP 可通过兴奋 P2Y 受体激活氯通道而产生与细胞容积调节相关的Cl- 电流。     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

Chloride channels involve in hydrogen peroxide-induced apoptosis of PC12 cells

Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H₂O₂)-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H₂O₂ activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H₂O₂ elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1 h and induced apoptosis of most PC12 cells tested in 24 h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H₂O₂-induced high membrane permeability and cell shrinkage, suppressed H₂O₂-activated chloride currents and protected PC12 cells from apoptosis induced by H₂O₂. The results suggest that chloride channels may contribute to H₂O₂-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.     

Ion channels are linked to differentiation in keratinocytes

Summary In vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca²⁺. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca²⁺, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca²⁺ and activated by physiological concentrations of Ca²⁺. We also found a 35 pS Cl^– channel whose open probability increased with depolarization. Finally, a 70 pS K⁺ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl^– channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K⁺ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.This work was supported in part by a National Institutes of Health grant K08 AR01853-03 and a National Science Foundation grant DCB-9009915 (to T.M.M.); National Institutes of Health Research Career Development Award K04 ARO 1803 and AR 39031 (to R.R.I.) and a National Institutes of Health grant GM-44840 (to P.A.P.).     

细胞松弛素B和鬼笔环肽对梨花粉管钙离子浓度和尖端钙离子通道的影响

应用激光共聚焦显微镜和全细胞膜片钳技术研究了微丝骨架解聚剂细胞松弛素B(CB)和稳定剂鬼笔环肽(PD)对梨花粉管细胞内钙离子浓度动态变化和尖端质膜上钙离子通道的影响。结果显示:CB处理能促进花粉管内胞质钙离子[Ca2+]i浓度增加,同时还能激活质膜上的钙离子通道;而PD处理对花粉管内[Ca2+]i浓度及钙离子通道几乎没有影响。研究表明,微丝骨架的解聚激活了花粉管质膜上的钙离子通道,使得胞外钙离子大量流入,胞内钙离子浓度升高,从而抑制花粉管生长。