Inactivation of human sodium channels and the effect of tocainide

The inactivation of the sodium channels in human medulloblastoma cells was investigated with the whole-cell recording technique. The potential dependence of inactivation (“inactivation curve”) was determined by imposing a series of prepulses of varying amplitude on the membrane potential and measuring the maximum sodium current flowing after each prepulse at the test potential of -20 mV. The time dependence of inactivation was investigated by determining inactivation curves with prepulses of variable duration. A prolongation of the prepulse increased the degree of inactivation, even when the prepulse duration was much greater than the time constant for fast inactivation. This is explained by the existence of two additional states of “intermediate” inactivation of the sodium channel, the transition to which is slower than that to the state of fast inactivation and faster than that to the state of slow inactivation. The antiarrhythmic drug tocainide had no effect on fast inactivation, but a strong effect on intermediate inactivation. This explains the use dependence of this drug. The reaction model given by Chiu (1977) for the transitions from the open into the closed state of inactivation and vice versa is extended.     

人骨髓间充质干细胞L型钙离子通道的研究

目的:研究分离培养的人骨髓间充质干细胞(hMSCs)膜表面L型钙离子通道的表达。方法:采用密度梯度离心、贴壁筛选及单克隆培养法获得hMSCs,利用流式细胞分析、免疫荧光法鉴定,应用全细胞膜片钳技术记录hMSCs膜表面L钙离子通道的变化。结果:体外分离、培养出高度同源性的具有独特细胞免疫学表型hMSCs;40%的hMSCs表达钙离子电流,峰值电流(ICaPeak)在+20～+30mV为(102.67±19.06)pA,此电流可被Cd2+(20μmolL)阻断。结论:在未分化的hMSCs上L型钙离子通道表达较少,说明L型钙离子通道表达的增多可能是hMSCs定向分化的条件之一,调节hMSCs膜表面L型钙离子通道的表达将有助于促进其定向分化。     

Expression of recombinant calcium channels support secretion in a mouse pheochromocytoma cell line

We have characterized a recently established mouse pheochromocytoma cell line (MPC 9/3L) as a useful model for studying neurotransmitter release and neuroendocrine secretion. MPC 9/3L cells express many of the proteins involved in Ca2+-dependent neurotransmitter release but do not express functional endogenous Ca2+-influx pathways. When transfected with recombinant N-type Ca2+ channel subunits alpha1B,beta2a,alpha2delta (Cav2.2), the cells expressed robust Ca2+ currents that were blocked by omega-conotoxin GVIA. Activation of N-type Ca2+ currents caused rapid increases in membrane capacitance of the MPC 9/3L cells, indicating that the Ca2+ influx was linked to exocytosis of vesicles similar to that reported in chromaffin or PC12 cells. Synaptic protein interaction (synprint) sites, like those found on N-type Ca2+ channels, are thought to link voltage-dependent Ca2+ channels to SNARE proteins involved in synaptic transmission. Interestingly, MPC 9/3L cells transfected with either LC-type (alpha1C, beta2a, alpha2delta, Cav1.2) or T-type (alpha1G, beta2a, alpha2delta, Cav3.1) Ca2+ channel subunits, which do not express synprint sites, expressed appropriate Ca2+ currents that supported rapid exocytosis. Thus MPC 9/3L cells provide a unique model for the study of exocytosis in cells expressing specific Ca2+ channels of defined subunit composition without complicating contributions from endogenous channels. This model may help to distinguish the roles that different Ca2+ channels play in Ca2+-dependent secretion.     

Store-operated calcium channels

In electrically nonexcitable cells, Ca(2+) influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and proliferation, and apoptosis. The major Ca(2+) entry pathway in these cells is the store-operated one, in which the emptying of intracellular Ca(2+) stores activates Ca(2+) influx (store-operated Ca(2+) entry, or capacitative Ca(2+) entry). Several biophysically distinct store-operated currents have been reported, but the best characterized is the Ca(2+) release-activated Ca(2+) current, I(CRAC). Although it was initially considered to function only in nonexcitable cells, growing evidence now points towards a central role for I(CRAC)-like currents in excitable cells too. In spite of intense research, the signal that relays the store Ca(2+) content to CRAC channels in the plasma membrane, as well as the molecular identity of the Ca(2+) sensor within the stores, remains elusive. Resolution of these issues would be greatly helped by the identification of the CRAC channel gene. In some systems, evidence suggests that store-operated channels might be related to TRP homologs, although no consensus has yet been reached. Better understood are mechanisms that inactivate store-operated entry and hence control the overall duration of Ca(2+) entry. Recent work has revealed a central role for mitochondria in the regulation of I(CRAC), and this is particularly prominent under physiological conditions. I(CRAC) therefore represents a dynamic interplay between endoplasmic reticulum, mitochondria, and plasma membrane. In this review, we describe the key electrophysiological features of I(CRAC) and other store-operated Ca(2+) currents and how they are regulated, and we consider recent advances that have shed insight into the molecular mechanisms involved in this ubiquitous and vital Ca(2+) entry pathway.     

Neurotrophin regulation of sodium and calcium channels in human neuroblastoma cells

Neurotrophins, acting through tyrosine kinase family genes, are essential for neuronal differentiation. The expression of tyrosine kinase family genes is prognostic in neuroblastoma, and neurotrophins reduce proliferation and induce differentiation, indicating that neuroblastomas are regulated by neurotrophins. We tested the effects of nerve growth factor and brain-derived neurotrophic factor on Na(+) and Ca(2+) currents, using the whole-cell patch-clamp technique, in human neuroblastoma NB69 cells. Control cells exhibited a slow tetrodotoxin-resistant (IC(50)=98 nM) Na(+) current and a high-voltage-activated Ca(2+) current. Exposure to nerve growth factor (50 ng/ml) and/or brain-derived neurotrophic factor (5 ng/ml) produced the expression of a fast tetrodotoxin-sensitive (IC(50)=10 nM) Na(+) current after day 3, and suppressed the slow tetrodotoxin-resistant variety. The same type of high-voltage-activated Ca(2+) current was expressed in control and treated cells. The treatment increased the surface density of both Na(+) and Ca(2+) currents with time after plating, from 17 pA/pF at days 3-5 and 1-5 to 34 and 30 pA/pF after days 6-10, respectively. Therefore, both nerve growth factor and brain-derived neurotrophic factor, acting through different receptors of the tyrosine kinase family and also possibly the tumor necrosis factor receptor-II, were able to regulate differentiation and the expression of Na(+) and Ca(2+) channels, partially reproducing the modifications induced by diffusible astroglial factors.We show that neurotrophins induced differentiation to a neuronal phenotype and modified the expression of Na(+) and Ca(2+) currents, partially reproducing the effects of diffusible astroglial factors.     

钙库操纵性钙通道在人循环纤维细胞中的表达及功能

 目的: 研究钙库操纵性钙通道(store-operated calcium channels,SOCC)相关功能蛋白ORAI1-3和STIM1-2在人循环纤维细胞(circulating fibrocytes)中的表达及SOCC对人循环纤维细胞分化的影响。方法: 采集健康人外周静脉血,分离出单个核细胞,体外培养分化为循环纤维细胞。采用RT-PCR和real-time PCR检测循环纤维细胞中ORAI1-3及STIM1-2的mRNA表达情况,并检测SOCC抑制剂对循环纤维细胞分化的影响。结果: Real-time PCR检测结果显示ORAI1-3和STIM1-2 mRNA在循环纤维细胞中有较高的表达水平,并且SOCC抑制剂SKF-96365对循环纤维细胞分化具有明显的抑制作用。结论: SOCC表达于循环纤维细胞中,并且影响循环纤维细胞的分化。     

The gating and conductance properties of Cav3.2 low-voltage-activated T-type calcium channels

Calcium channels are essential for excitation-contraction coupling and pacemaker potentials in cardiac muscle cells. Whereas L-type Ca(2+) channels have been extensively studied, T-type channels have been poorly characterized in cardiac myocytes. We describe here the functional properties of recombinant Ca(V)3.2 T-type Ca(2+) channels expressed in mammalian cell lines. The T-type Ca(2+) current showed a rapid activation and an inactivation phase in response to depolarization, and it displayed a window current over the voltage range from -60 to -40 mV in 1 to 10 mM external Ca(2+). Barium (Ba(2+)) and strontium (Sr(2+)) permeate the channel with similar activation kinetics. On the other hand, monovalent cations, Li(+) and Na(+), permeate the T-type Ca(2+) channel more easily than the L-type Ca(2+) channel. The permeability order of the Ca(V)3.2 T-type Ca(2+) channel among monovalent and divalent cations was determined as Ba(2+)>Mn(2+)>Ca(2+)>Sr(2+)>Li(+1)>Na(+) with the permeability order of 1.39:1.25:1.00:0.95:0.55:0.29. The ionic conductance sequence for cations relative to calcium was Sr(2+)>Ba(2+)>Ca(2+)>Li(+1)>Mn(2+)>Na(+) with the conductance ratio of 1.39:1.21:1.00:0.40:0.23:0.11. The permeation profile of manganese (Mn(2+)) is complex. Mn(2+) permeates the Ca(2+) channel with a permeability similar to Ca(2+) or Ba(2+), but with a much smaller current density, resulting in a much smaller conductance. The properties relating to progression and recovery from inactivation in the Ca(V)3.2 channel are substantially identical with either Ca(2+) or Ba(2+) as the charge carrier.     

Biophysical properties of CaV1.3 calcium channels in gerbil inner hair cells

The Ca²⁺ current ( I _Ca) in prehearing and adult inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, is mainly carried by L-type (Ca_V1.3) Ca²⁺ channels. I _Ca in immature and adult IHCs triggers the release of neurotransmitter onto auditory afferent fibres in response to spontaneous action potentials (APs) or graded receptor potentials, respectively. We have investigated whether the biophysical properties of I _Ca vary between low- and high-frequency IHCs during cochlear development and whether its inactivation influences cellular responses. I _Ca was recorded from gerbil IHCs maintained near physiological recording conditions. The size of I _Ca in adult IHCs was about a third of that in immature cells with no apparent difference along the cochlea at both stages. The activation kinetics of I _Ca were significantly faster in high-frequency IHCs, with that of adult cells being more rapid than immature cells. The degree of I _Ca inactivation was similar along the immature cochlea but larger in high- than low-frequency adult IHCs. This inactivation was greatly reduced with barium but not affected by changing the intracellular buffer (BAPTA instead of EGTA). Immature basal IHCs showed faster recovery of I _Ca from inactivation than apical cells allowing them to support a higher AP frequency. I _Ca in adult IHCs was more resistant to progressive inactivation following repeated voltage stimulation than that of immature cells. This suggests that adult IHCs are likely to be suited for sustaining rapid and repeated release of synaptic vesicles, which is essential for sound encoding.     

Functional impact of alternative splicing of human T-type Cav3.3 calcium channels

Low-voltage-activated T-type (Cav3) Ca2+ channels produce low-threshold spikes that trigger burst firing in many neurons. The CACNA1I gene encodes the Cav3.3 isoform, which activates and inactivates much more slowly than the other Cav3 channels. These distinctive kinetic features, along with its brain-region-specific expression, suggest that Cav3.3 channels endow neurons with the ability to generate long-lasting bursts of firing. The human CACNA1I gene contains two regions of alternative splicing: variable inclusion of exon 9 and an alternative acceptor site within exon 33, which leads to deletion of 13 amino acids (Delta33). The goal of this study is to determine the functional consequences of these variations in the full-length channel. The cDNA encoding these regions were cloned using RT-PCR from human brain, and currents were recorded by whole cell patch clamp. Introduction of the Delta33 deletion slowed the rate of channel opening. Addition of exon 9 had little effect on kinetics, whereas its addition to Delta33 channels unexpectedly slowed both activation and inactivation kinetics. Modeling of neuronal firing showed that exon 9 or Delta33 alone reduced burst firing, whereas the combination enhanced firing. The major conclusions of this study are that the intracellular regions after repeats I and IV play a role in channel gating, that their effects are interdependent, suggesting a direct interaction, and that splice variation of Cav3.3 channels provides a mechanism for fine-tuning the latency and duration of low-threshold spikes.     

Voltage-sensitive calcium channels in a human small-cell lung cancer cell line

Pancrazio , J.J., Oie , H.K. & Kim , Y.I. 1992. Voltage-sensitive calcium channels in a human small-cell lung cancer cell line. Acta Physiol Scand 144 , 463468. Received 1 October 1 991 , accepted 24 October 1991. ISSN 0001–6772. Departments of Biomedical Engineering and Neurology, University of Virginia Health Sciences Center, and the National Cancer Institute, Navy Medical Oncology Branch, Naval Hospital Bethesda, USA. Utilizing the whole-cell patch-clamp method we assessed the Ca²⁺ current (I_ca) in well-established cell lines from human small-cell carcinoma (SCC) of the lung, NCI-H209 and NCI-H187. The Ca²⁺ current was readily observed in H209 tumour cells (90% of the cells tested), whereas H187 tumour cells only occasionally expressed Ca^z+ channels (26% of the cells tested). H209 Ca²⁺ current was evoked by potentials greater than -30 mV and exhibited partial inactivation over the duration of a 40 ms command potential. This inward current was unchanged by alteration of the holding potential from - 80 to - 40 mV and the activation phase of the Ca²⁺ current was best fitted by Hodgkin-Huxley m(t)² kinetics. H209 Ca²⁺ current was reduced over 80% by verapamil (100 μM), whereas w-conotoxin (5 μM) appeared to be without effect. In contrast, H209 Ca²⁺ current was rapidly abolished by nifedipine (10 μm), strongly suggesting the presence of L-type Ca²⁺ channels. Voltage-gated Ca²⁺ channels may be important to the secretion of ectopic hormones and the etiology and pathogenesis of Lambert-Eaton syndrome, an autoimmune disorder of the motor nerve terminal in which autoantibodies directed against voltage-gated Ca²⁺ channels are produced.     

Amazing T-type calcium channels: updating functional properties in health and disease

T-type Ca²⁺ channels have gained, 15 years after cloning, an immense interest as novel players in very unexpected cell functions, and its many relations to diseases have been discovered. This special issue provides a state-of-the-art overview on novel functional properties of T-type Ca²⁺ channels, unexpected cellular functions, and most importantly will also summarizes and review the involvement of this “tiny, transient” type of Ca²⁺ channels in several diseases. It is tried to bridge the gap between molecular biophysical properties of T-type Ca²⁺ channels and diseases providing finally a translational view on this amazing ion channel.     

Altered regulation of calcium channels and exocytosis in single human pheochromocytoma cells

We established primary cultures of human pheochromocytoma chromaffin cells. We then tried to find what mechanism of their secretory apparatus could be altered to produce the massive release of catecholamines into the circulation and the subsequent hypertensive crisis observed in patients suffering this type of tumor. Their whole-cell Ca2+ channel currents could be pharmacologically separated into components similar to those found in normal human adrenal chromaffin cells: 20% L-type, 30% N-type, and 50% P/Q-type Ca2+ channels. However, modulation of the channels by exogenous or endogenous ATP and opioids, via a G-protein membrane-delimited pathway, was deeply altered; some cells having no modulation or very little modulation alternated with others having normal modulation. This may be the cause of the uncontrolled secretory response, measured amperometrically at the single-cell level. Some cells secreted for long time periods and were insensitive to nifedipine (L-type channel blocker) or to omega-conotoxin MVIIC (N/P/Q-type channel blocker), while others were highly sensitive to nifedipine and partially sensitive to omega-conotoxin MVIIC. Alteration of the autocrine/paracrine modulation of Ca2+ channels may lead to indiscriminate Ca2+ entry and exacerbate catecholamine release responses in human pheochromocytoma cells.     

Biochemical properties of recombinant human and mouse N-acetylglutamate synthase

N-Acetylglutamate synthase (NAGS, EC 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG) from glutamate and acetylcoenzyme A. NAG is an obligatory activator of carbamylphosphate I (CPSI), the first and a rate limiting enzyme of ureagenesis. The enzymatic activity of NAGS increases in the presence of arginine. Since the level of NAGS activity depends on the concentrations of two amino acids, glutamate and arginine, and it supplies the essential cofactor for CPSI, NAGS may play an important role in the regulation of ureagenesis. The amino acid sequences of human and mouse NAGS consist of three regions with different degrees of conservation: the mitochondrial targeting signal (MTS), the variable domain, and the conserved domain. Removal of the MTS results in mature NAGS (NAGS-M) while removal of the MTS and the variable domain results in conserved NAGS (NAGS-C). The biochemical properties of purified recombinant human and mouse NAGS-M and NAGS-C were determined in this study with the goal of better understanding the role of the variable domain in NAGS function. The activity of all four proteins doubled in the presence of arginine, while the affinities for substrates changed less than two fold. The turnover numbers of NAGS-C are double those of NAGS-M proteins. Processing of NAGS-M to form NAGS-C results in an enzyme with higher catalytic activity and could play a role in the regulation of NAG production, CPSI function, and urea synthesis.     

Giga-seal formation alters properties of sodium channels of human myoballs

The influence of giga-seal formation on the properties of the Na⁺ channels within the covered membrane patch was investigated with a whole-cell pipette and a patch pipette applied to the same cell. Current kinetics, current/voltage relation and channel densities were determined in three combinations: (i) voltage-clamping and current recording with the whole-cell pipette, (ii) voltage-clamping with the whole-cell pipette and current recording with the patch pipette and, (iii) voltage-clamping and current recording with the patch pipette. The Hodgkin-Huxley (1952) parameters _m and _h were smaller for the patch currents than for the whole cell, and the h curve was shifted in the negative direction. The channel density was of the order of 10 times smaller. All effects were independent of the extracellular Ca²⁺ concentration. The capacitive current generated in the patch by the whole-cell Na⁺ current and its effect on the transmembrane voltage of the patch were evaluated. The kinetic parameters of the Na⁺ channels in the patch did not depend on whether the voltage was clamped with the whole-cell pipette or the patch pipette. Thus, the results are not due to spurious voltage.