培养大鼠颈上神经节单烟碱受体通道及突触电流的膜片钳研究

在培养１─２周的大鼠颈上神经节交感神经元标本上,用膜片钳技术记录了单烟碱受体通道电流及胆碱能突触电流,并分析了它们之间的关系。单烟碱受体通道至少有三种亚导状态,即１５ｐＳ,２７ｐＳ,３８ｐＳ,其中以２７ｐＳ最常见。通道有两种开放模式,即单个短促开放与长串开放。对应的平均开放时间分别为τ＿１＝１．７１ｍｓ,τ＿２＝１２．２４ｍｓ。对自发突触电流的分析表明,其下降相的衰减时间常数（τ＝１５．７±１ｍｓ）与上述长串开放的持续时间相当,提示在突触传递过程中,突触前末梢释放的ＡＣｈ引起了突触后神经元烟碱受体通道的长串开放。     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

电压依赖性钾通道与人类神经性疾病

电压依赖性钾通道是钾通道超家族中成员最多,最为复杂的亚家族,主要包括Kvα亚单位和辅助亚单位两部分,其中快速失活A型通道和毒蕈碱敏感的M通道已被大量研究,它们广泛分布于神经系统,主要参与各种生理和病理作用,如膜兴奋性的产生,神经递质的释放,神经元细胞的增殖和退化,以及神经网络的信号传递等。目前发现Kv通道亚型或亚单位的突变与学习和记忆的损伤,共济失调,癫痫,神经性耳聋等一些神经性疾病的产生有关。     

Effects of reagents modifying carboxyl groups on the gating current of the myelinated nerve fiber

Summary K⁺ channels in inside-out patches from hamster insulin tumor (HIT) cells were studied using the patch-clamp technique. HIT cells provide a convenient system for the study of ion channels and insulin secretion. They are easy to culture, form gigaohm seals readily and secrete insulin in response to glucose. The properties of the cells changed with the passage number. For cell passage numbers 48 to 56, five different K⁺-selective channels ranging from 15 to 211 pS in symmetrical 140mm KCl solutions were distinguished. The channels were characterized by the following features: a channel with a conductance (in symmetrical 140mm KCl solutions) of 210 pS that was activated by noncyclic purine nucleotides and closed by H⁺ ions (pH=6.8); a 211 pS channel that was Ca²⁺-activated and voltage dependent; a 185 pS channel that was blocked by TEA but was insensitive to quinine or nucleotides; a 130 pS channel that was activated by membrane hyperpolarization; and a small conductance (15 pS) channel that was not obviously affected by any manipulation. As determined by radioimmunoassay, cells from passage number 56 secreted 917±128 ng/mg cell protein/48 hr of insulin. In contrast, cells from passage number 77 revealed either no channel activity or an occasional nonselective channel, and secreted only 29.4±8.5 ng/mg cell protein/48 hr of insulin. The nonselective channel found in the passage 77 cells had a conductance of 25 pS in symmetrical 140mm KCl solutions. Thus, there appears to be a correlation between the presence of functional K⁺ channels and insulin secretion.     

Blockade of L-type voltage-gated Ca channel inhibits ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.     

Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K _i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K _i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn²⁺ ions, in particular the αS583C mutant showing a K _i for Zn²⁺of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K _i, the later mutation generating a Ca²⁺blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na⁺ions at an external Na⁺binding site preventing ion permeation through the channel pore.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.     

Metabolic flux analysis gives an insight on verapamil induced changes in central metabolism of HL-1 cells

Verapamil has been shown to inhibit glucose transport in several cell types. However, the consequences of this inhibition on central metabolism are not well known. In this study we focused on verapamil induced changes in metabolic fluxes in a murine atrial cell line (HL-1 cells). These cells were adapted to serum free conditions and incubated with 4 μM verapamil and [U-¹³C₅] glutamine. Specific extracellular metabolite uptake/production rates together with mass isotopomer fractions in alanine and glutamate were implemented into a metabolic network model to calculate metabolic flux distributions in the central metabolism. Verapamil decreased specific glucose consumption rate and glycolytic activity by 60%. Although the HL-1 cells show Warburg effect with high lactate production, verapamil treated cells completely stopped lactate production after 24 h while maintaining growth comparable to the untreated cells. Calculated fluxes in TCA cycle reactions as well as NADH/FADH₂ production rates were similar in both treated and untreated cells. This was confirmed by measurement of cell respiration. Reduction of lactate production seems to be the consequence of decreased glucose uptake due to verapamil. In case of tumors, this may have two fold effects; firstly depriving cancer cells of substrate for anaerobic glycolysis on which their growth is dependent; secondly changing pH of the tumor environment, as lactate secretion keeps the pH acidic and facilitates tumor growth. The results shown in this study may partly explain recent observations in which verapamil has been proposed to be a potential anticancer agent. Moreover, in biotechnological production using cell lines, verapamil may be used to reduce glucose uptake and lactate secretion thereby increasing protein production without introduction of genetic modifications and application of more complicated fed-batch processes.     

Evaluation of swimming performance for fish passage of longnose dace Rhinichthys cataractae using an experimental flume

The swimming performance of longnose dace Rhinichthys cataractae, the most widely distributed minnow (Cyprinidae) in North America, was assessed in relation to potential passage barriers. The study estimated passage success, maximum ascent distances and maximum sprint speed in an open‐channel flume over a range of water velocities and temperatures (10·7, 15·3 and 19·3° C). Rhinichthys cataractae had high passage success (95%) in a 9·2 m flume section at mean test velocities of 39 and 64 cm s^–1, but success rate dropped to 66% at 78 cm s^–1. Only 20% of fish were able to ascend a 2·7 m section with a mean velocity of 122 cm s^–1. Rhinichthys cataractae actively selected low‐velocity pathways located along the bottom and corners of the flume at all test velocities and adopted position‐holding behaviour at higher water velocities. Mean volitional sprint speed was 174 cm s^–1 when fish volitionally sprinted in areas of high water velocities. Swimming performance generally increased with water temperature and fish length. Based on these results, fishways with mean velocities <64 cm s^–1 should allow passage of most R. cataractae. Water velocities >100 cm s^–1 within structures should be limited to short distance (<1 m) and structures with velocities ≥158 cm s^–1 would probably represent movement barriers. Study results highlighted the advantages of evaluating a multitude of swimming performance metrics in an open‐channel flume, which can simulate the hydraulic features of fishways and allow for behavioural observations that can facilitate the design of effective passage structures.