Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca⁺⁺ and inhibited by Mg⁺⁺, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol. 258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.Abbreviations used: PT: permeability transition; PTP: permeability transition pore; MMC: mitochondrial megachannel; IMAC: inner membrane anion channel. P_A: permeability of ion A. CSP: Cyclosporin A.     

Voltage activation of heart inner mitochondrial membrane channels

The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above ±60mV [Kinnallyet al., Biochem. Biophys. Res. Commun. 176, 1183–1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin Aare likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.     

Voltage dependent sodium currents in cultured rat cerebellar granule cells

Sodium currents were studied in granule cells dissociated from rat cerebellum. Macroscopic currents were recorded using the patch-clamp technique. Sudium currents, which are TTX sensitive, reached a maximum peak value of 0.42±0.08 pA/m² at 18.4±2.2 mV (n=6). Activation and inactivation kinetics and steady-state properties were described in terms of Hodgkin and Huxley, parameters. The properties of sodium channels in cultured rat cerebellar granule cells are very similar to those reported for various neural preparations.     

An efficient isolation procedure of Ca-tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies

A simple procedure which provides a large yield of isolated ferret ventricular myocytes is described. The enzymatic dissociation was performed by perfusion of the whole heart with the "Langendorff method" at 37 degrees C, without an incubation period. Special attention was given to the period of perfusion with Ca-free or low-calcium containing solutions and to the proportion of both collagenase and elastase used. The viability and calcium tolerance of the isolated cells were tested by ultrastructural and electrophysiological studies. Photo-microscopy showed that 60 to 80% of the isolated cells had an elongated shape (18 microns in diameter, 150 microns in length) and did not beat spontaneously in normal Tyrode solution. The morphological and ultrastructural integrity of these cells was shown in SEM by their smooth surface with regularly spaced T-tubule openings and in TEM by the regular distribution of the transverse tubular system, mitochondrium and sarcomeres. Using the whole-cell patch-clamp technique, they had a resting membrane potential of -72 mV, two types ("Purkinje like" and "ventricular like") of action potentials could be elicited and they were correctly affected by well-known modulators of calcium channels. This technique was successfully applied to the rat heart and could be used for heart dissociation of small mammals. It can simultaneously provide isolated cells of different regions of the heart and can be easily and routinely used by any investigator.     

川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

本工作旨在研究川芎嗪对猪冠状动脉平滑肌细胞钾通道的作用,为阐明其扩张冠状动脉血管的机制提供实验依据。采用膜片钳细胞贴附式和内面向外式记录方式观察川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductance Ca2+- activated potassium channels,BKCa channels)的作用,分别用蛋白激酶A(protein kinase A,PKA)抑制剂H-89和蛋白激酶G (protein kinase G,PKG)抑制剂KT-5823处理细胞,再观察川芎嗪对BKCa通道作用的变化。结果表明在研究的0．73-8．07 mmol/L浓度范围,川芎嗪可以剂量依赖性地激活BKCa通道,使通道的开放概率从(0．01±0．003)增加到(0．03±0．01)-(．21± 0．18)(P<0．01,n=10),使通道平均关闭时间从(732．33±90．67)ms降低到(359．67±41．30)-(2．96±0．52)ms(P<0．01, n=10)。川芎嗪的这种激活作用在浴液游离钙离子浓度接近0 mmol/L时也存在。PKA的特异性抑制剂H-89(3 μmol/L)和 PKG的特异性抑制剂KT-5823(1 μmol/L)对川芎嗪激活BKCa通道的作用无影响。以上结果提示:川芎嗪能直接激活冠状动脉平滑肌BKCa通道,这种作用可能是川芎嗪扩张冠状动脉血管的一种重要机制。     

Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC(50) value for ACh of 19 microm. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 microm ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.     

AGS3蛋白对吗啡慢性处理大鼠前额叶皮层神经元瞬时外向钾电流的影响

目的:研究吗啡对大鼠皮层神经元瞬时外向钾电流(IA)的影响,并在此基础上,用G蛋白信号转导激活因子3(AGS3)的抗体阻断AGS3作用,观察其对瞬时外向钾电流(IA)的影响,从而探讨AGS3蛋白在吗啡成瘾中的机制。方法:采用全细胞膜片钳技术记录IA;吗啡对神经元IA电流密度-电压曲线(I-V曲线)的影响;在全细胞构型下,观察三种不同浓度AGS3抗体对吗啡处理大鼠前额叶皮层神经元IA的影响。结果:吗啡能引起大鼠皮质神经元IA增强;当膜电位+55mV时,10-3μg/L、10-2μg/L、10-1μg/L三种不同浓度的AGS3抗体作用于吗啡处理的神经元,10-3μg/L对电流密度的抑制没有显著差异;10-2μg/L、10-1μg/L的抗体能显著抑制吗啡引起的电流密度升高,有统计学差异。结论:吗啡能引起神经元I的增强,AGS3蛋白在成瘾机体中参与了对I通道进行调节的信号转导通路。     

一种适合膜片钳单通道记录的大鼠DRG神经元急性分离方法

目的：建立一种适合膜片钳单通道记录的脊髓背根神经节神经元急性分离方法。方法：用酶消化和机械分离相结合的方法急性分离大鼠DRG神经元。结果：用本方法分离的DRG细胞容易形成较高的封接电阻（〉5GΩ）,降低了噪音干扰,可记录到pA级的单通道电流。结论：本方法急性分离的DRG神经元适合单通道膜片钳实验研究。