The proton ladder,a static mechanism for ion/proton coports and counterports

An ion/proton counterport is formed simply by locating a chain of ionizable residues connected by a proton conducting path near a passive ion pore which spans the membrane. The electric coupling between the ion in transit through the pore and the residues can ensure that for each ion passing through the pore in one direction a proton is driven along the chain of ionizable residues (the proton ladder) in the same or in the opposite direction. The mechanism is symmetrical in that a trans-membrane ion gradient may drive protons against their electrochemical potential gradient or a proton gradient may drive ions against theirs. The mechanism is applicable to cation or anion channels and to coports or counterports. No mechanical motion is required other than the motion of the ions and the protons. Monte Carlo computer simulations are performed on the model and its predicted properties are listed. The new type of counterport model is compared with currently used models. Offprint requests to: D. T Edmonds     

The CLC ‘chloride channel’ family: revelations from prokaryotes (Review)

Members of the CLC ‘chloride channel’ family play vital roles in a wide variety of physiological settings. Research on prokaryotic CLC homologues provided long-anticipated high-resolution structures as well as the unexpected discovery that some CLCs are not chloride channels, but rather are proton-chloride antiporters. Hence, CLCs encompass two functional classes of transport proteins once thought to be fundamentally different from one another. In this review, we discuss the structural features and molecular mechanisms of CLC channels and antiporters. We focus on ClC-0, the most thoroughly studied CLC channel, and ClC-ec1, the prokaryotic antiporter of known structure. We highlight some striking similarities between these CLCs and discuss compelling questions that remain to be addressed. Prokaryotic CLCs will undoubtedly continue to shed light upon this understudied family of proteins.     

A unifying concept for ion translocation by retinal proteins

First, halorhodopsin is capable of pumping protons after illumination with greenand blue light in the same direction as chloride. Second, mutated bacteriorhodopsin where the proton acceptor Asp85 and the proton donor Asp96 are replaced by Asn showed proton pump activity after illumination with blue light in the same direction as wildtype after green light illumination. These results can be explained by and are discussed in light of our new hypothesis: structural changes in either molecule lead to a change in ion affinity and accessibility for determining the vectoriality of the transport through the two proteins.     

Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Inhibition of cytochrome b563-oxidation by triorganotins in spinach chloroplasts

The triorganotin compounds triphenyltin chloride and tributyltin chloride have been known as inhibitors of the transmembrane proton channel forming F₀-domain of ATPases at micromolar concentrations. We show that these compounds at higher concentrations (10–100 µM) also inhibit uncoupled electron transport in chloroplasts within the low potential chain of the cytochrome bf complex. They cause high levels of transiently reduced cytochrome b563 as they decelerate the reoxidation process in flash illuminated chloroplasts. At the same time they slow down the flash induced slow electrogenic step generated at the cytochrome bf complex. The inhibitory effect of triphenyltin chloride on cytochrome b563 turnover in chloroplasts is comparable to that of the Q_n-inhibitor MOA-stilbene, with even less side effects on the high potential chain. Studies on the isolated bf complex suggest different binding sites for triorganotins and the quinone analogue type Q_n-inhibitors. The results are interpreted within the framework of the modified Q-cycle model by a putative organotin sensitive proton translocating site which enables proton transfer from the outer aqueous face of the membrane to the hydrophobic quinone reduction site within the complex. Hence, cytochrome b563 oxidation and plastoquinone reduction may be inhibited as a consequence of proton transfer being suppressed by triorganotins. In analogy, the previously described inhibitory effect of Val/K⁺ at the n-side of the cytochrome bf complex [Klughammer and Schreiber (1993) FEBS 336: 491–495] may be rationalised by binding of the cyclic depsipeptide at the entrance of the proton path to the Q_n-site.     

三苯基锡,二环己基碳二亚胺和寡霉素在叶绿体中的作用特点

作用于质子通道的抑制剂(TPT,DCCD和OM)对与叶绿体中质子跨膜传导失去联系的甲醇活化的类囊体膜上或离体的CF1—ATP酶活性无抑制作用,其主要作用部位在质子通道CF_0上。TPT和 DCCD能恢复残缺膜积贮H_in~ 、重建△pH和△ψ,使其电子流从解联状态回到复与叶绿体基础电子流相一致的水平。低浓度TPT即可部分恢复残缺膜的DLE,而DCCD需较高的浓度。低浓度TPT对叶绿体DLE有明显促进作用,而高浓度TPT和 DCCD有抑制作用。OM对这些功能都没有明显影响。推测OM可能作用于CF_0与CF_1的连接处,TPT的作用部位可能与光系统Ⅱ的水氧化质子释放部位有联系。     

Review. Proton-coupled gating in chloride channels

The physiologically indispensable chloride channel (CLC) family is split into two classes of membrane proteins: chloride channels and chloride/proton antiporters. In this article we focus on the relationship between these two groups and specifically review the role of protons in chloride-channel gating. Moreover, we discuss the evidence for proton transport through the chloride channels and explore the possible pathways that the protons could take through the chloride channels. We present results of a mutagenesis study, suggesting the feasibility of one of the pathways, which is closely related to the proton pathway proposed previously for the chloride/proton antiporters. We conclude that the two groups of CLC proteins, although in principle very different, employ similar mechanisms and pathways for ion transport.     

Identification of Three Distinct Phylogenetic Groups of CAX Cation/Proton Antiporters

Ca²⁺/cation antiporter (CaCA) proteins are integral membrane proteins that transport Ca²⁺ or other cations using the H⁺ or Na⁺ gradient generated by primary transporters. The CAX (for CAtion eXchanger) family is one of the five families that make up the CaCA superfamily. CAX genes have been found in bacteria, Dictyostelium, fungi, plants, and lower vertebrates, but only a small number of CAXs have been functionally characterized. In this study, we explored the diversity of CAXs and their phylogenetic relationships. The results demonstrate that there are three major types of CAXs: type I (CAXs similar to Arabidopsis thaliana CAX1, found in plants, fungi, and bacteria), type II (CAXs with a long N-terminus hydrophilic region, found in fungi, Dictyostelium, and lower vertebrates), and type III (CAXs similar to Escherichia coli ChaA, found in bacteria). Some CAXs were found to have secondary structures that are different from the canonical six transmembrane (TM) domains–acidic motif-five TM domain structure. Our phylogenetic tree indicated no evidence to support the cyanobacterial origin of plant CAXs or the classification of Arabidopsis exchangers CAX7 to CAX11. For the first time, these results clearly define the CAX exchanger family and its subtypes in phylogenetic terms. The surprising diversity of CAXs demonstrates their potential range of biochemical properties and physiologic relevance. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: David Guttman]     

Ion channel blockade attenuates aggregated alpha synuclein induction of microglial reactive oxygen species: relevance for the pathogenesis of Parkinson's disease

Brain mononuclear phagocyte (perivascular macrophage and microglia, MG) inflammatory neurotoxins play a principal role in the pathogenesis of Parkinson's disease; chief among these are reactive oxygen species (ROS). We posit that aggregated, misfolded and oxidized alpha-synuclein (a major constituent of Lewy bodies), released or secreted from dying dopaminergic neurons, induces microglial ROS production that is regulated by ion channels and as such affects disease progression. To address this hypothesis, we performed patch clamp recordings of outward ionic currents in murine microglia and characterized their links to ROS production during alpha-synuclein stimulation. Aggregated nitrated alpha-synuclein induced ROS production in a dose-dependent manner that was inhibited by voltage-gated potassium current blockade, and to a more limited degree, by chloride current blockade. Interestingly, ROS produced in MG primed with tumor necrosis factor alpha and activated with phorbol myristate acetate was attenuated by voltage-gated potassium current blockade and more completely by chloride current blockade. In contrast, amyloid beta or cell membrane extract failed to induce microglial ROS production. Similar results were obtained using bone marrow-derived macrophages. The association of ROS production with specific plasma membrane ion currents provides a link between regulation of microglial ion transport and oxygen free radical production. Understanding these linkages may lead to novel therapeutics for Parkinson's disease where modulation of redox-related stress may slow disease progression.     

Anti-interleukin 6 (IL-6) receptor antibody suppresses Castleman's disease like symptoms emerged in IL-6 transgenic mice

Transgenic mice carrying human IL-6 cDNA fused with a murine major histocompatibility class-I promoter (H-2L(d)) were serially administered with anti-interleukin-6 receptor (IL-6R) monoclonal antibody (mAb), MR16-1, from the age of 4 weeks to estimate its efficacy on a variety of disorders developed in these mice, most of which are similar to the disorders associated with Castleman's disease. In the control mice treated with isotype-matched mAb, a massive and multiple IgG1 plasmacytosis, mesangial proliferative glomerulonephritis, leukocytosis, thrombocytosis, anemia and abnormalities of blood chemical parameters have developed in accordance with the elevation of serum IL-6, and 50% of mice have died of renal failure by 18 weeks of age. In contrast, the treatment with MR16-1 prevented all these symptoms and prolonged the lifetime of the majority of the mice. Thus, the constitutive overexpression of IL-6 caused various disorders, and the treatment with anti-IL-6R mAb completely prevented from these symptoms. These results clearly confirm that IL-6 indeed plays an essential role in the pathogenesis of a variety of disorders. Furthermore, anti-IL-6R mAb could provide novel therapy for Castleman's disease and MR16-1 should be a useful tool to estimate therapeutic potential of IL-6 antagonists in a variety of murine models for human disease.     

叶绿体中电色效应慢上升相的起源,质子释放过程及细胞色素b_6、f的氧化还原反应

P515吸收变化中的慢上升相(P515s),可用Tris,NH_4Cl或尼日利亚菌素,加在低盐浓度的叶绿体中形成。在叶绿体和叶片样品中,不管有无P515s,都有明显的细胞色素b_6和f的氧化还原变化。在有解联剂存在下,中性红染料可测到膜内质醌醇氧化释放质子的过程,释放速度与P515s形成的速度接近。高盐浓度中暗适应的叶绿体,释放H~+的速度比P515s形成的速度快,其中质醌醇释放H~+的速度只有1～2 ms。在接近生理条件下(无外加供受体、抑制剂),P515s的起源与细胞色素b_6、f的氧化还原反应造成的电子跨膜流动无直接联系,而质醌醇释放H~+的过程及随后的侧向扩散,可能是P515s形成的直接起因。     

Magnetic fields at resonant conditions for the hydrogen ion affect neurite outgrowth in PC-12 cells: A test of the ion parametric resonance model

PC-12 cells primed with nerve growth factor (NGF) were exposed to sinusoidal extremely-low-frequency (ELF) magnetic fields (MFs) selected to test the predictions of the ion parametric resonance (IPR) model under resonance conditions for a single ion (hydrogen). We examined the field effects on the neurite outgrowth (NO) induced by NGF using three different combinations of flux densities of the parallel components of the AC MF (B_ac) and the static MF (B_dc). The first test examined the NO response in cells exposed to 45 Hz at a B_dc of 2.96 μT with resonant conditions for H⁺ according to the model. The B_ac values ranged from 0.29 to 4.11 μT root-mean-square (rms). In the second test, the MF effects at off-resonance conditions (i.e., no biologically significant ion at resonance) were examined using the frequency of 45 Hz with a B_dc of 1.97 μT and covering a B_ac range between 0.79 and 2.05 μT rms. In the third test, the AC frequency was changed to 30 Hz with the subsequent change in B_dc to 1.97 μT to tune for H⁺ as in the first test. The B_ac values ranged from 0.79 to 2.05 μT rms. After a 23 h incubation and exposure to the MF in the presence of NGF (5 ng/ml), the NO was analyzed using a stereoscopic microscope. The results showed that the NGF stimulation of neurite outgrowth (NSNO) was affected by MF combinations over most of the B_ac exposure range generally consistent with the predictions of the IPR model. However, for a distinct range of B_ac where the IPR model predicted maximal ionic influence, the observed pattern of NSNO contrasted sharply with those predictions. The symmetry of this response suggests that values of B_ac within this distinct range may trigger alternate or additional cellular mechanisms that lead to an apparent lack of response to the MF stimulus. © 1996 Wiley-Liss, Inc.     

Tumor metabolism,cancer cell transporters,and microenvironmental resistance

Cancer cells reprogram their metabolic machineries to enter into permanent glycolytic pathways. The full reason for such reprogramming takes place is unclear. However, this metabolic switch is not made in vain for the lactate that is generated and exported outside cells is reused by other cells. This results in the generation of a pH gradient between the low extracellular pH that is acidic (pHe) and the higher cytosolic alkaline or near neutral pH (pHi) environments that are tightly regulated by the overexpression of several pumps and ion channels (e.g. NHE-1, MCT-1, V-ATPase, CA9, and CA12). The generation of this unique pH gradient serves as a determining factor in defining “tumor fitness”. Tumor fitness is the capacity of the tumor to invade and metastasize due to its ability to reduce the efficiency of the immune system and confer resistance to chemotherapy. In this article, we highlight the importance of tumor microenvironment in mediating the failure of chemotherapeutic agents.     

The energetics of Cl− active transport inChara

Summary The rate of Cl^– influx in intactChara was inhibited whenever the ATP concentration was reduced by application of metabolic inhibitors. In perfused cells, however, a net influx of Cl^– against its electrochemical gradient could be observed in the absence of ATP. Addition of ATP to the perfusion medium slightly stimulated Cl^– influx in one experiment but had no effect in another. Addition of ADP, NADH or metabolic inhibitors did not alter the influx rate. Consideration of the potential energy gradients across theChara plasmalemma in the perfused state leads to the conclusion that Cl^– influx occurs by cotransport with H⁺ or OH^–.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.