Differences in the binding modes of phytochelatin to cadmium (II) and Zinc(II) ions

An ¹H NMR (nuclear magnetic resonance) spectroscopic structural analysis of Cd²⁺ complexes formed with the pentapeptide phytochelatin, (NH₃)⁺−(ψ-Glu-Cys)₂−Gly−COO−(PC₂), at a pH of 7.5 showed that the two thiol groups of the Cys residues and either the carbonyl or amide group of the peptide bond between Glu1 and Cys1 act as possible donor groups in the complexes at Cd²⁺/PC₂ ratios up to 0.4. As the ratio increases, the carboxylate group of Glu2 and either the carbonyl or amide group of the peptide bond between Cys1 and Glu2 comes to serve as a donor group. The manner in which Cd²⁺ forms complexes with PC₂ is distinctly different from Zn²⁺ and might account for the role of phytochelatin in Cd²⁺ detoxification. Electron absorption spectrometry demonstrated that the Cd²⁺ complexes are coordinated in a tetrahedral fashion by four thiol groups and that several sulfur atoms might bridge Cd²⁺ ions, resulting in the formation of polynuclear complexes. This contrasts with Zn²⁺ complex formation, which consists exclusively of a 1:1 complex.     

Metal binding to ligands: cadmium complexes with glutathione revisited

We studied the interaction of gamma-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) with cadmium ions (Cd(2+)) by first performing classical potentiometric pH titration measurements and then turning to additional spectroscopic methods. To estimate the residual concentrations of free cadmium, we studied the competition of glutathione with a Cd(2+)-sensitive dye, either an absorbing dye (murexide) or a fluorescent one (FluoZin-1), and consistent results were obtained with the two dyes. In KCl-containing Tes, Mops, or Tris buffer at pH 7.0 to 7.1 and 37 degrees C (and at a total Cd(2+) concentration of 0.01 mM), results suggest that free cadmium concentration is halved when the concentration of glutathione is approximately 0.05 mM; this mainly reflects the combined apparent dissociation constant for the Cd(glutathione) 1:1 complex under these conditions. To identify the other complexes formed, we used far-UV spectroscopy of the ligand-to-metal charge transfer absorption bands. The Cd(glutathione)(2) 1:2 complex predominated over the 1:1 complex only at high millimolar concentrations of total glutathione and not at low submillimolar concentrations of total glutathione. The apparent conditional constants derived from these spectroscopy results made it possible to discriminate between sets of absolute constants that would otherwise have simulated the pH titration data similarly well in this complicated system. Related experiments showed that although the Cl(-) ions in our media competed (modestly) with glutathione for binding to Cd(2+), the buffers we had chosen did not bind Cd(2+) significantly under our conditions. Our experiments also revealed that Cd(2+) may be adsorbed onto quartz or glass vessel walls, reducing the accuracy of theoretical predictions of the concentrations of species in solution. Lastly, the experiments confirmed the rapid kinetics of formation and dissociation of the UV-absorbing Cd(glutathione)(2) 1:2 complexes. The methods described here may be useful for biochemists needing to determine conditional binding constants for charge transfer metal-ligand complexes under their own conditions.     

重金属Cd2+和Cu2+胁迫下泥蚶消化酶活性的变化

为了探讨重金属Cd²⁺和Cu²⁺胁迫对泥蚶消化酶活性的影响,运用酶学分析的方法,按《渔业水质标准》（GB 11607）规定的Cd²⁺、Cu²⁺最高限量值的1、2、5、10倍设置重金属离子Cd²⁺、Cu²⁺浓度及其组合,研究了在重金属Cd²⁺、Cu²⁺胁迫下,30d内泥蚶3种消化酶活性的变化规律。结果表明：与空白对照组相比,在重金属Cd²⁺、Cu²⁺或其组合的胁迫下,较低浓度组泥蚶的淀粉酶活性实验前期增强（即被诱导）,实验后期减弱（即被抑制）,较高浓度组泥蚶的淀粉酶活性从实验一开始就减弱,并保持在较低水平,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合Cu²⁺ > （Cd²⁺+Cu²⁺）组合 > Cd²⁺;泥蚶脂肪酶的活性实验前期增强,实验后期转为微减弱或减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺;泥蚶胃蛋白酶的活性实验前期增强,且活性呈现升高-降低-再升高-再降低的变化,实验后期分别表现微增强、微减弱和减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺。可见：环境中的Cd²⁺和Cu²⁺对泥蚶的消化酶活性起着明显的影响作用。     

镉离子污染条件下微生物群落中细菌与藻类的相互作用

【背景】水体微生物有着丰富的多样性,不同种类的微生物之间的相互作用对水体生态系统的组成结构与功能具有重要影响。水体内的藻类与某些微生物可以发生多种相互作用,然而人们对逆境条件下的菌藻有益相互作用尚缺乏深入研究。【目的】为了研究镉对水体微生物群落的影响以及镉胁迫下菌藻之间可能的相互作用。【方法】本研究运用了基于16S rRNA基因的高通量测序技术,分析在不同Cd~(2+)条件下微生物群落结构的变化,利用微生物相互作用网络分析菌藻之间可能发生的相互作用。【结果】通过分离培养筛选出了与集胞藻PCC6803互作抗Cd~(2+)的关键细菌Y9菌株。【结论】研究结果表明Y9菌株属于Phyllobacteriaceae科,与微生物群落组成和微生物互作网络的分析结果相符。本研究为探索水体环境中微生物种间相互作用、菌藻互作抗Cd~(2+)的生态效应提供参考依据。     

Factorial design for the optimization of enzymatic detection of cadmium in aqueous solution using immobilized urease from vegetable waste

Free as well as alginate immobilized urease was utilized for detection and quantitation of cadmium (Cd2+) in aqueous samples. Urease from the seeds of pumpkin (Cucumis melo), being a vegetable waste, was extracted and purified to apparent homogeneity (Sp. Activity 353 U/mg protein; A280/A260=1.12) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization and no leaching of the enzyme was found over a period of 15 days at 4 degrees C. Urease catalyzed urea hydrolysis by both soluble and immobilized enzyme revealed a clear dependence on the concentration of Cd2+. The inhibition caused by Cd2+ was non-competitive (Ki=1.41 x 10(-5) M). The time dependent inhibition both in the presence and in absence of Cd2+ ion revealed a biphasic inhibition in the activity. A Response Surface Methodology (RSM) for the parametric optimization of this process was performed using two-level-two-full factorial (2(2)), central composite design (CCD). The regression coefficient, regression equation and analysis of variance (ANOVA) was obtained using MINITAB 15 software. The predicted values thus obtained were closed to the experimental value indicating suitability of the model. In addition to this 3D response surface plot and isoresponse contour plot were helpful to predict the results by performing only limited set of experiments.     

河豚毒素和Cd2+对Zn2+诱发爆发波放电的影响

本文用微电极细胞内电位记录、通道阻断剂和放射性同位素等技术发现,锌离子可诱发爆发波放电(BD),钠通道阻断剂——河豚毒素对BD无效应,而钙通道阻断剂——Ca²⁺则可使BD消失,Cd²⁺可使[⁶⁵Zn²⁺]_i量减少。以上结果说明,Zn²⁺诱发BD的产生机理很可能是Zn²⁺代替Ca²⁺通过钙通道进入胞内引起的。     

Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars

Effects of cadmium (Cd(2+)) on photosynthetic and antioxidant activities of maize (Zea mays L.) cultivars (3223 and 32D99) were investigated. Fourteen-day-old cultivar seedlings were exposed to different Cd concentrations [0, 0.3, 0.6 and 0.9mM Cd(NO(3))(2).4H(2)O] for 8 days. The results of chlorophyll fluorescence indicated that different levels of Cd affected photochemical efficiency in 3223 much more than that in 32D99. In parallel, the level of Cd at 0.9mM caused oxidative damage but did not indicate cessation of PSII activity of the cultivars; plant death was not observed at highly toxic Cd levels. Additionally, the increase in Cd concentration caused loss of chlorophylls and carotenoid and membrane damage in both cultivars, but greater membrane damage was observed in 3223 than in 32D99. Depending on Cd accumulation, a significant reduction in dry biomass was observed in both cultivars at all Cd concentrations. The accumulation of Cd was higher in roots than in leaves for both cultivars. Nevertheless, cultivar 3223 transferred more Cd from roots to leaves than did 32D99. On the other hand, our results suggest that there were similar responses in SOD, APX and GR activities with increasing Cd concentrations for both cultivars. However, POD activity significantly increased at highly toxic Cd levels in 32D99. This result may be regarded as an indication of better tolerance of the Z. mays L. cultivar 32D99 to Cd contamination.     

Movements near the gate of a hyperpolarization-activated cation channel

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Like the related depolarization-activated K+ channels (Kv channels), HCN channels use an intracellular activation gate to regulate access to an inner cavity, lined by the S6 transmembrane regions, which leads to the selectivity filter near the extracellular surface. Here we describe two types of metal interactions with substituted cysteines in the S6, which alter the voltage-controlled movements of the gate. At one position (L466), substitution of cysteine in all four subunits allows Cd2+ ions at nanomolar concentration to stabilize the open state (a "lock-open" effect). This effect depends on native histidines at a nearby position (H462); the lock-open effect can be abolished by changing the histidines to tyrosines, or enhanced by changing them to cysteines. Unlike a similar effect in Kv channels, this effect depends on a Cd2+ bridge between 462 and 466 in the same subunit. Cysteine substitution at another position (Q468) produces two effects of Cd2+: both a lock-open effect and a dramatic slowing of channel activation-a "lock-closed" effect. The two effects can be separated, because the lock-open effect depends on the histidine at position 462. The novel lock-closed effect results from stabilization of the closed state by the binding of up to four Cd2+ ions. During the opening conformational change, the S6 apparently moves from one position in which the 468C cysteines can bind four Cd2+ ions, possibly as a cluster of cysteines and cadmium ions near the central axis of the pore, to another position (or flexible range of positions) where either 466C or 468C can bind Cd2+ in association with the histidine at 462.     

Cd2+、Hg2+对菱幼苗生长及其超氧化物歧化酶、过氧化物酶活性的影响

利用不同浓度Ｃｄ＾２＋、Ｈｇ＾２＋处理菱幼苗,研究重金属离子对菱生长、超氧化物歧化酶（ＳＯＤ）、过氧化物酶（ＰＯＤ）活性的影响,比较了Ｃｄ＾２＋、Ｈｇ＾２＋对同一植物的毒性差异,Ｃｄ＾２＋、Ｈｇ＾＾２＋各处理浓度与均抑制菱幼苗生长,使叶绿素含量下降,Ｃｄ＾２＋的抑制作用比Ｈｇ＾２＋的作用明显。Ｃｄ＾２＋、Ｈｇ＾２＋对ＳＯＤ、ＰＯＤ活性有不同的影响效果;Ｃｄ＾２＋处理最艉地ＳＯＤ、ＰＯＤ活性升高,但     

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane

Summary Two-dimensional crystalline arrays of Ca²⁺-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na₃VO₄ in a Ca²⁺-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3-diethyl-2,2-thiadicarbocyanine (Di–S–C₂(5)) to monitor inside positive or inside negative membrane, potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca²⁺-ATPase, while negative potential disrupts preformed Ca²⁺-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca²⁺-ATPase.     

Bridging the gaps in 3D structure of the inositol 1,4,5-trisphosphate receptor-binding core

Calcium concentration is strictly regulated in all cells. The inositol 1,4,5-trisphosphate receptor (IP(3)R), which forms a homotetrameric Ca2+ release channel in the endoplasmic reticulum, is one of the key molecules responsible for this regulation. The opening of this channel requires binding of two intracellular messengers, which are inositol 1,4,5-trisphosphate (IP(3)) and Ca2+. To promote the Ca2+-channel gating and release from the endoplasmic reticulum, IP(3) binds to the amino-terminal region of IP(3)R. Recently, the crystal structure of IP(3)R-binding core in complex with its ligand was presented [I. Bosanac, J.R. Alattia, T.K. Mai, J. Chan, S. Talarico, F.K. Tong, K.I. Tong, F. Yoshikawa, T. Furuichi, M. Iwai, T. Michikawa, K. Mikoshiba, M. Ikura, Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700; I. Bosanac, H. Yamazaki, T. Matsu-ura, T. Michikawa, K. Mikoshiba, M. Ikura, Crystal structure of the ligand-binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor, Mol. Cell 17 (2005) 193-203]. The space positions of residues 289-301 (segment A), 320-350 (segment B), 373-386 (segment C), and 529-545 (segment D) were not determined by the X-ray crystallography. To bridge these gaps, the computer modeling of physiologically meaningful low-energy 3D structures of the segments A-D of the inositol 1,4,5-trisphosphate receptor has been carried out by using a hierarchical conformational search algorithm combining two approaches: knowledge-based homology modeling and ab initio conformational search strategy. The structure analysis suggests a Ca2+-binding site of high affinity formed by residues 296-335, several low-energy regular secondary structure units within the segment B, and a number of hinge regions within the segments A-D, important for the receptor functioning.     

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.     

雷公藤红素抑制Cd2+诱导的神经毒性北大核心CSCD

镉(cadmium,Cd)是环境中常见的一种重金属,Cd^(2+)可以通过穿透血脑屏障,产生神经毒性,从而诱发各种神经退行性疾病,雷公藤红素是雷公藤的一种有效成分,具有抗癌、抗炎等一系列药理作用,本文探究雷公藤红素对Cd^(2+)诱导的相应神经毒性的影响作用。通过细胞增殖实验、细胞膜完整性实验、细胞形态实验探索了Cd^(2+)对小胶质细胞HMC3活力的影响;通过一氧化氮(NO)检测实验、脂质过氧化(malondialdehyde,MDA)检测实验、蛋白免疫印迹实验分析了Cd^(2+)的神经毒性以及雷公藤红素对Cd^(2+)诱导的相应神经毒性的影响。结果表明:与对照组相比,当Cd^(2+)浓度达到40μmol/L时,对HMC3细胞增殖抑制率为(57.17±8.23)%(P<0.01,n=5),继续增大Cd^(2+)浓度,细胞活性将进一步降低;当Cd^(2+)浓度达到40μmol/L以上时,HMC3的细胞膜明显受到破坏,并且破坏作用与浓度呈剂量依赖性关系;随着Cd^(2+)浓度的增加,细胞形态开始变化,贴壁效果变差。Cd^(2+)使HMC3细胞释放的NO量显著增加,而雷公藤红素能够有效地抑制Cd^(2+)诱导的HMC3细胞NO的释放;Cd^(2+)使HMC3细胞脂质过氧化水平显著增加,加入10^(-7) mol/L雷公藤红素后,MDA的释放量显著减少;Cd^(2+)会使p-PI3K蛋白含量增加,而加入了雷公藤红素(10^(-7)、10^(-6) mol/L)后,p-PI3K蛋白和p-AKT蛋白的激活均被抑制,从而抑制了细胞凋亡。综上所述,雷公藤红素能够抑制Cd^(2+)诱导的小胶质细胞毒性,从而起到神经保护作用。     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel.

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101). However, the ZD7288 molecule is larger than the Na(+) or K(+) ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd(2+) applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd(2+) was applied to opened channels, with an apparent blocking rate of approximately 3 x 10(5) M(-1)s(-1). The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd(2+) access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd(2+) inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd(2+) could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd(2+) inhibition. Thus, Cd(2+) escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.     

Cadmium inhibits plasma membrane calcium transport

Summary The interaction of Cd²⁺ with the plasma membrane Ca²⁺-transporting ATPase of fish gills was studied. ATP-driven Ca²⁺-transport in basolateral membrane (BLM) vesicles was inhibited by Cd²⁺ with anI ₅₀ value of 3.0nm at 0.25 m free Ca²⁺ using EGTA, HEEDTA and NTA to buffer Ca²⁺ and Cd²⁺ concentrations. The inhibition was competitive in nature since theK _0.5 value for Ca²⁺ increased linearly with increasing Cd²⁺ concentrations while theV _max remained unchanged. The Ca²⁺ pump appeared to be calmodulin dependent, but we conclude that the inhibition by Cd²⁺ occurs directly on the Ca²⁺ binding site of the Ca²⁺-transporting ATPase and not via the Ca²⁺-binding sites of calmodulin. It is suggested that Cd²⁺-induced inhibition of Ca²⁺-transporting enzymes is the primary effect in the Cd²⁺ toxicity towards cells followed by several secondary effects due to a disturbed cellular Ca²⁺ metabolism. Our data illustrate that apparent stimulatory effects of low concentrations of Cd²⁺ on Ca²⁺-dependent enzymes may derive from increased free-Ca²⁺ levels when Cd²⁺ supersedes Ca²⁺ on the ligands.     

Techno-economic and environmental approaches of Cd2+ adsorption by olive leaves (Olea europaea L.) waste

Abstract

In this study, the techno-economic approach of olive leaves (Olea europaea L.) wastes for the removal of Cd²⁺ from aqueous solutions was demonstrated. The adsorption process was illustrated regarding batch experiments and scanning electron microscopy, energy dispersive X-ray, and Fourier-transform infrared characterization. The optimum pH and contact time were 6.6 and 123?min, respectively, giving Cd²⁺ removal efficiencies of 94.9% at C_o = 50?mg/L and 81.5% at C_o = 100?mg/L. The monolayer adsorption capacity of the Langmuir isotherm model was 32.6?mg/g (R² = 0.97). The adsorption mechanisms might be related to (a) ion exchange with cations (e.g., K⁺, Na⁺, and Ca²⁺), (b) formation of cadmium chloride complexes, (c) interaction with oxygen-containing functional groups, (d) physical agglomeration in the pore surface, and (e) precipitation interaction using inorganic minerals (i.e., carbonates, phosphates, and silicates). The total cost of the adsorption process for the treatment of ions-containing wastewater was 0.038 $USD/m³. Assuming a benefit-cost of tertiary treated water as 0.044 $USD/m³, the adsorption system could attain a payback period of 5.7?years. This period was shorter than the lifetime of the capital investment (i.e., 10?years), and hence, the project would be economically feasible for an application.     

A spectroscopic study of the interaction of the VO2+ cation with the two components of chondroitin sulfate

The interaction of the vanadyl (IV) cation with N-acetyl-D-galactosamine, D-galactosamine, and D-glucuronic acid has been investigated by electron absorption spectroscopy at different mental to ligand ratios and pH values. In the case of D-glucuronic acid, a more detailed study was undertaken, using differential IR spectroscopy in solution. The results show that the cation interacts with the two nitrogenated molecules only at higher pH values, generating 2∶1 lig-and to metal complexes in which coordination occurs through two pairs of deprotonated OH groups of the rings. In the case of D-glucuronic acid, the IR-measurements allowed a wider insight into the structural characteristics of the complexes generated in acidic media. The involvement of the glycosidic oxygen atom in coordination, is suggested at pH=3.     

P-loop Flexibility in Na+ Channel Pores Revealed by Single- and Double-cysteine Replacements

Replacement of individual P-loop residues with cysteines in rat skeletal muscle Na⁺ channels (SkM1) caused an increased sensitivity to current blockade by Cd²⁺ thus allowing detection of residues lining the pore. Simultaneous replacement of two residues in distinct P-loops created channels with enhanced and reduced sensitivity to Cd²⁺ block relative to the individual single mutants, suggesting coordinated Cd²⁺ binding and cross-linking by the inserted sulfhydryl pairs. Double-mutant channels with reduced sensitivity to Cd²⁺ block showed enhanced sensitivity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one another to within less than 3.5 Å. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd²⁺ with a single residue in another P-loop. These results suggest that, on the time-scale of Cd²⁺ binding to mutant Na⁺ channels, P-loops show a high degree of flexibility.