Alkali metal ion selectivity of the hemocyanin channel

Summary The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K⁺ measured from biionic potentials were: NH ₄ ⁺ (1.52)>Rb⁺ (1.05)>K⁺ (1.0)>Cs⁺ (0.89)>Na⁺ (0.81)>Li⁺ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K⁺, Rb⁺, NH ₄ ⁺ , Cs⁺, Na⁺ and Li⁺, respectively. Current-voltage curves for the different monovalent cations were measured and described using a threebarrier model previously used to explain the voltage dependence of the instantaneous channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH ₄ ⁺ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.     

The barium site in a potassium channel by x-ray crystallography

X-ray diffraction data were collected from frozen crystals (100 degrees K) of the KcsA K(+) channel equilibrated with solutions containing barium chloride. Difference electron density maps (F(barium) - F(native), 5.0 A resolution) show that Ba(2+) resides at a single location within the selectivity filter. The Ba(2+) blocking site corresponds to the internal aspect (adjacent to the central cavity) of the "inner ion" position where an alkali metal cation is found in the absence of the blocking Ba(2+) ion. The location of Ba(2+) with respect to Rb(+) ions in the pore is in good agreement with the findings on the functional interaction of Ba(2+) with K(+) (and Rb(+)) in Ca(2+)-activated K(+) channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.     

Structure modeling of the acetylcholine receptor channel and related ligand gated channels

In this model-building study a model for the pore of the acetylcholine receptor channel is proposed. The pore is formed by five -helices of the M2 segment where three rings of hydrophilic side chains point into the channel lumen. This model is in agreement with most experimental data like photolabeling, drug affinity studies, single channel conductivity measurements and cryo electron microscopy known about this channel.This study predicts a strong coupling of the motion of the ions in the channel to that of the charged and highly hydrophilic amino acid side chains at the channel wall. Due to the negative net-charge in the pore more than a single cation may occupy the pore region. The resulting strong local electric fields make the commonly used constant field approximation obsolete for this type of ion channel.     

A comparison of sodium channel kinetics in the squid axon,the frog node and the frog node with BTX using the “silent gate” model

In this paper it is shown that the very different kinetics measured for the rise of the sodium current which follows a depolarization of the membrane in the squid giant axon, the frog node and the frog node treated with Batrachotoxin may be accurately predicted using only the measured equilibrium and static characteristics for the three preparations and the kinetics measured for the gating charge transfer.The kinetic predictions follow the use of the silent gate model for ion channel gating. The model is electrostatic and its chief assumptions are that the channel gate, called here the N-system, has fast kinetics and responds to the gating charge that transfers but not directly to the trans-membrane voltage applied. Because channel gating, corresponding here to the motion of the N-system, does not change its energy in the trans-membrane applied electric field the gating is electrically silent as far as gating charge transfer measurement is concerned. However the probability of gating rises with the quantity of gating charge that transfers due to the electrostatic interaction between the N-system and the gating charge, redistributed under the influence of the applied trans-membrane electric field. With these assumptions the kinetics of sodium channel gating are predictable using only the static and equilibrium characteristics of gating charge and channel activation measured as a function of membrane voltage, and the kinetics of the gating charge transfer. Because of the fast kinetics assumed for the N-system the predicted kinetics are the same for channels with any number of equivalent and independent N-systems or gates acting in parallel.The model predictions for sodium permeability kinetics are compared in detail with those recently measured for the frog node treated with Batrachotoxin and excellent agreement is obtained.     

桂西北石灰土土壤有机碳矿化对外源有机物质和碳酸钙的响应

为明确外源有机物质和无机碳酸盐对桂西北石灰土土壤有机碳矿化的影响,加深对土壤有机碳周转特征的认识,本文以广西环江县喀斯特地区的棕色石灰土、黑色石灰土和地带性红壤(对照)为研究对象,进行为期100 d的室内培养试验[对照(无外源物添加,CK)、添加14C-稻草(S)、添加Ca14CO3(C)],并对土壤呼吸释放的CO2及14C-CO2含量进行测定.结果表明:培养100 d后,外源物的添加均明显促进了红壤、棕色和黑色石灰土有机碳的矿化,外源14C-稻草和Ca14 CO3对上述土壤有机碳矿化的激发效应分别为28.7％、46.2％ 、15.5％和127.0％ 、175.3％、100.1％;土壤表观累积矿化量中外源Ca14CO3的贡献率分别为40.4％、48.4％、19.6％;土壤类型和添加物及两者间的交互作用均对土壤有机碳矿化的激发效应、土壤表观累积矿化量中外源物的贡献、土壤有机碳的矿化速率、土壤有机碳累积矿化量/率有显著影响.因此,外源有机物质和碳酸钙的添加改变了土壤有机碳的矿化特征,对于含碳酸盐的石灰土,研究土壤有机碳矿化、周转规律,评估其对大气CO2的影响必须考虑无机碳酸盐的贡献.     

基于FLUXNET观测数据与VPM模型的森林生态系统光合作用关键参数优化及验证

生态系统总初级生产力(GPP)是全球生态系统碳循环研究的重要组成部分.植被最大光能利用率(ε_max)是陆地生态系统GPP模拟的关键参数.本文基于植被光合模型(VPM)和全球通量网(FLUXNET)40个站点(179条站点年数据)的涡度相关通量观测数据,采用单因素轮换法对VPM模型进行参数敏感性分析,并利用交叉验证法对全球森林生态系统的光合作用关键参数进行优化和验证.结果表明:森林生态系统GPP模型受ε_max、光合最高温度(T_max)以及光合最适温度(T_opt)的影响最大;优化后的ε_max在不同植被类型之间存在明显差异,介于0.05～0.08 μmol CO₂·μmol^-1 PAR,常绿阔叶林>常绿针叶林>混交林>落叶阔叶林;优化后的森林生态系统T_max为38～48 ℃,T_opt为18～22 ℃;利用分植被类型优化后的模型关键参数,VPM模型可较好地模拟全球主要森林生态系统GPP的季节和年际变化.     

离子注入水稻愈伤组织提高农杆菌转化效率的初步研究

通过ＧＵＳ报告基因表达的组织化学染色分析,我们发现低能氮离子束注入水稻愈伤组织可显著提高根癌农杆菌介转基因效率,这为克服单子叶植物对根癌农杆菌不敏感性、提高遗传转化效率的研究提供了一条新的途径。     

Extracellular sodium interacts with the HERG channel at an outer pore site

Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.     

Identification and experimental verification of a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels

Studies of bacterial ion channels have provided significant insights into the structure-function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.     

Na~+、K~+离子通道阻滞剂对离体大鼠黄体细胞孕酮生成的影响

本工作用二种离子通道阻断剂四乙胺(TEA)和河豚毒素(TTX)来研究 Na~+、K~+通道的改变对大鼠黄体细胞孕酮生成的影响。10~(-3)mol/L 的 TEA 或 TTX 均使孕酮分泌量显著增加,而这种促进效应可被酪氨酸(Tyr)完全阻断。Tyr 对 TEA 或 TTX 与 hCG 联合所引起的孕酮分泌也有抑制作用。上述实验说明跨黄体细胞内外的 K~+和 Na~+浓度差与孕酮分泌有关。