Bacterial ion channels and their eukaryotic homologues.

Due to the relative ease of obtaining their crystal structures, bacterial ion channels provide a unique opportunity to analyse structure and function of their eukaryotic homologues. This review describes prokaryotic channels whose structures have been determined. These channels are KcsA, a bacterial homologue of eukaryotic potassium channels, MscL, a bacterial mechanosensitive ion channel and ClC0, a prokaryotic homologue of the eukaryotic ClC family of anion-selective channels. General features of their structure and function are described with a special emphasis on the advantages that these channels offer for understanding the properties of their eukaryotic homologues. We present amino-acid sequences of eukaryotic proteins related in their primary sequences to bacterial mechanosensitive channels. The usefulness of bacterial mechanosensitive channels for the studies on general principles of mechanosensation is discussed.     

The venerable inveterate invertebrate TRP channels

The transient receptor potential (TRP) superfamily is subdivided into four main classes of cation channels, TRPC, TRPV, TRPM and TRPN, each of which includes members in worms, flies, mice and humans. While the biophysical features of many of the mammalian channels have been described, relatively little is known concerning the biological roles of these channels. Forward genetic screens in Drosophila melanogaster and Caenorhabditis elegans have led to the identification of the founding members of each of these four subfamilies. Moreover, phenotypic analyses of invertebrate mutants have contributed greatly to our understanding of the roles of TRP proteins. A recurring theme is that many of these proteins function in sensory signaling processes ranging from vision to olfaction, osmosensation, light touch, social feeding, and temperature- and mechanically-induced nociception. In addition, at least one invertebrate TRP protein is required for cell division. As many of these functions may be conserved among the mammalian TRPs, the invertebrate TRPs offer valuable genetic handles for characterizing the functions of these cation channels in vivo.     

Ion channels formed by chemical analogs of gramicidin A.

Channel-forming peptides such as gramicidin A offer the opportunity to study the relationship between chemical structure and transport properties of an ion channel. This article summarizes a number of recent experiments with chemical analogs and derivatives of gramicidin A using artificial lipid bilayer membranes. The introduction of negative charges near the channel mouth leads to an increase in the cation transport rate. Hybrid channels consisting of a neutral and a negatively charged or of a positively and a negatively charged half-channel may be formed. The current-voltage characteristic of these hybrid channels exhibits a pronounced asymmetry.Experiments with charged derivatives of gramicidin A have been used in order to distinguish between different structural models of the dimeric channel; these studies strongly support Urry's model of a single-stranded, head-to-head associated helical dimer. In a further set of experiments gramicidin analogs with modified amino acid sequence were studied. It was found that a single substitution (tryptophan replaced by phenylalanine) leads to marked changes in the conductance of the channel. Analogs with a simplified amino acid sequence such as (L-Trp-D-Leu)7-L-Trp or L-Trp-Gly-(L-Trp-D-Leu)6-L-Trp are able to form cation permeable channels with similar properties as gramicidin A.     

Can rice field channels contribute to biodiversity conservation in Southern Brazilian wetlands?

Conservation of species in agroecosystems has attracted attention. Irrigation channels can improve habitats and offer conditions for freshwater species conservation. Two questions from biodiversity conservation point of view are: 1) Can the irrigated channels maintain a rich diversity of macrophytes, macroinvertebrates and amphibians over the cultivation cycle? 2) Do richness, abundance and composition of aquatic species change over the rice cultivation cycle? For this, a set of four rice field channels was randomly selected in Southern Brazilian wetlands. In each channel, six sample collection events were carried out over the rice cultivation cycle (June 2005 to June 2006). A total of 160 taxa were identified in irrigated channels, including 59 macrophyte species, 91 taxa of macroinvertebrate and 10 amphibian species. The richness and abundance of macrophytes, macroinvertebrates and amphibians did not change significantly over the rice cultivation cycle. However, the species composition of these groups in the irrigation channels varied between uncultivated and cultivated periods. Our results showed that the species diversity found in the irrigation channels, together with the permanence of water enables these man-made aquatic networks to function as important systems that can contribute to the conservation of biodiversity in regions where the wetlands were converted into rice fields. The conservation of the species in agriculture, such as rice field channels, may be an important alternative for biodiversity conservation in Southern Brazil, where more than 90% of wetland systems have already been lost and the remaining ones are still at high risk due to the expansion of rice production.     

Voltage-gated K+ channel modulators as neuroprotective agents

A manifestation in neurodegeneration is apoptosis of neurons. Neurons undergoing apoptosis may lose a substantial amount of cytosolic K⁺ through a number of pathways including K⁺ efflux via voltage-gated K⁺ (Kv) channels. The consequent drop in cytosolic [K⁺] relieves inhibition of an array of pro-apoptotic enzymes such as caspases and nucleases. Blocking Kv channels has been known to prevent neuronal apoptosis by preventing K⁺ efflux. Some neural diseases such as epilepsy are caused by neuronal hyperexcitability, which eventually may lead to neuronal apoptosis. Reduction in activities of A-type Kv channels and Kv7 subfamily members is amongst the etiological causes of neuronal hyperexcitation; enhancing the opening of these channels may offer opportunities of remedy. This review discusses the potential uses of Kv channel modulators as neuroprotective drugs.     

Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane

Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of -41 mV (SD 16) and -112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.     

Calcium regulation of tip growth: new genes for old mechanisms

We review the recent advances on Ca(2+) in tip-growing cells, with a special focus on pollen tubes. New genes for Ca(2+) pumps, channels and sensing proteins have been recently described, with special emphasis on cyclic nucleotide gated channels (CNGCs) and glutamate receptor-like channels (GLRs). We also review the current state of knowledge in what concerns Ca(2+) sensor and relay proteins, where the knowledge of the cell models is less advanced. While these newly described genes offer promise to a better understanding of the spatial and temporal patterns of Ca(2+) signalling that may be relevant for the formation of the phenotype, we discuss the necessity to investigate further links in the network downstream of the Ca(2+) signature, with a special need for mechanisms of feed-back that might render functional feed-back loops approachable by modelling and genetics. Given the available literature, we conclude on the need to investigate more on the role of two specific classes of proteins, the calcium binding protein kinases (CPKs) and the Calcineurin B-like proteins (CBLs) and their regulatory relationships to ion channels (summarized in Figure 3b).     

Engineering enzyme access tunnels

Enzymes are efficient and specific catalysts for many essential reactions in biotechnological and pharmaceutical industries. Many times, the natural enzymes do not display the catalytic efficiency, stability or specificity required for these industrial processes. The current enzyme engineering methods offer solutions to this problem, but they mainly target the buried active site where the chemical reaction takes place. Despite being many times ignored, the tunnels and channels connecting the environment with the active site are equally important for the catalytic properties of enzymes. Changes in the enzymatic tunnels and channels affect enzyme activity, specificity, promiscuity, enantioselectivity and stability. This review provides an overview of the emerging field of enzyme access tunnel engineering with case studies describing design of all the aforementioned properties. The software tools for the analysis of geometry and function of the enzymatic tunnels and channels and for the rational design of tunnel modifications will also be discussed. The combination of new software tools and enzyme engineering strategies will provide enzymes with access tunnels and channels specifically tailored for individual industrial processes.     

Apamin inhibits NO-induced relaxation of the spontaneous contractile activity of the myometrium from non-pregnant women

There is now considerable evidence for the involvement of K⁺ channels in nitric oxide (NO) induced relaxation of smooth muscles including the myometrium. In order to assess whether apamin-sensitive K⁺ channels play a role in NO – induced relaxation of the human uterus, we have studied the effect of specific blockers of these channels on the relaxation of myometrium from non-pregnant women. In vitro isometric contractions were recorded in uterine tissues from non-pregnant premenopausal women who had undergone hysterectomy. Apamin (10 nM) and scyllatoxin (10 nM) did not alter spontaneous myometrial contractions. However, 15-min pretreatment of the myometrium strips with apamin completely inhibited relaxation caused by diethylamine-nitric oxide (DEA/NO). The pretreatment with scyllatoxin significantly reduced (about 2.6 times) maximum relaxation of the strips induced by DEA/NO (p < 0.05). These results strongly suggest that, beside Ca²⁺ and voltage dependent charybdotoxin-sensitive (CTX-sensitive) K⁺ channels, apamin-sensitive K⁺ channels are also present in the human non-pregnant myometrium. These channels offer an additional target in the development of new tocolytic agents.     

A high-Na(+) conduction state during recovery from inactivation in the K(+) channel Kv1.5

Na(+) conductance through cloned K(+) channels has previously allowed characterization of inactivation and K(+) binding within the pore, and here we have used Na(+) permeation to study recovery from C-type inactivation in human Kv1.5 channels. Replacing K(+) in the solutions with Na(+) allows complete Kv1.5 inactivation and alters the recovery. The inactivated state is nonconducting for K(+) but has a Na(+) conductance of 13% of the open state. During recovery, inactivated channels progress to a higher Na(+) conductance state (R) in a voltage-dependent manner before deactivating to closed-inactivated states. Channels finally recover from inactivation in the closed configuration. In the R state channels can be reactivated and exhibit supernormal Na(+) currents with a slow biexponential inactivation. Results suggest two pathways for entry to the inactivated state and a pore conformation, perhaps with a higher Na(+) affinity than the open state. The rate of recovery from inactivation is modulated by Na(+)(o) such that 135 mM Na(+)(o) promotes the recovery to normal closed, rather than closed-inactivated states. A kinetic model of recovery that assumes a highly Na(+)-permeable state and deactivation to closed-inactivated and normal closed states at negative voltages can account for the results. Thus these data offer insight into how Kv1. 5 channels recover their resting conformation after inactivation and how ionic conditions can modify recovery rates and pathways.     

Intracellular detection assays for high-throughput screening

New optical assay methods promise to accelerate the use of living cells in screens for drug discovery. Most of these methods employ either fluorescent or luminescent read-outs and allow cell-based assays for most targets, including receptors, ion channels and intracellular enzymes. Furthermore, genetically encoded probes offer the possibility of custom-engineered biosensors for intracellular biochemistry, specifically localized targets, and protein—protein interactions.     

多不饱和脂肪酸对钾通道的调控作用及机理

多不饱和脂肪酸具有包括离子通道在内的众多作用靶点,通过作用于这些靶点,可以有效保护免疫系统、神经系统和心血管系统的功能,在一定程度上保护人体健康。电压门控钾离子通道家族K_V7通道和大电导钙离子激活的钾离子通道(BK_Ca)广泛表达于机体的各类组织中,具有重要的生理或病理功能。本综述围绕K_V7和BK_Ca通道,根据对已有报道的汇总,多不饱和脂肪酸可以增大K_V7和BK_Ca通道的电流幅值,其中对K_V7通道电流的影响主要是改变其电压依赖特性和最大电导值,而对BK_Ca通道电流的影响主要是改变其孔道区域关闭态的构象。此外,多不饱和脂肪酸对K_V7和BK_Ca通道功能的调节也会受到共表达的辅助亚基影响,但相关机制有待进一步阐明。深入理解多不饱和脂肪酸对K_V7和BK_Ca通道调节作用效果和分子机制,有助于全面理解K_V7和BK     

A STIMulating journey into optogenetic engineering

Genetically-encoded calcium actuators (GECAs) stemmed from STIM1 have enabled optical activation of endogenous ORAI1 channels in both excitable and non-excitable tissues. These GECAs offer new non-invasive means to probe the structure-function relations of calcium channels and wirelessly control the behavior of awake mice.     

Molecular dynamics simulations of the calmodulin-induced α-helix in the SK2 calcium-gated potassium ion channel

The family of small-conductance Ca²⁺-activated potassium ion channels (SK channels) is composed of four members (SK1, SK2, SK3, and SK4) involved in neuron-firing regulation. The gating of these channels depends on the intracellular Ca²⁺ concentration, and their sensitivity to this ion is provided by calmodulin (CaM). This protein binds to a specific region in SK channels known as the calmodulin-binding domain (CaMBD), an event which is essential for their gating. While CaMBDs are typically disordered in the absence of CaM, the SK2 channel subtype displays a small prefolded α-helical region in its CaMBD even if CaM is not present. This small helix is known to turn into a full α-helix upon CaM binding, although the molecular-level details for this conversion are not fully understood yet. In this work, we offer new insights on this physiologically relevant process by means of enhanced sampling, atomistic Hamiltonian replica exchange molecular dynamics simulations, providing a more detailed understanding of CaM binding to this target. Our results show that CaM is necessary for inducing a full α-helix along the SK2 CaMBD through hydrophobic interactions with V426 and L427. However, it is also necessary that W431 does not compete for these interactions; the role of the small prefolded α-helix in the SK2 CaMBD would be to stabilize W431 so that this is the case. In conclusion, our findings provide further insight into a key interaction between CaM and SK channels that is important for channel sensitivity to Ca²⁺.     

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.     

Sea urchin sperm cation-selective channels directly modulated by cAMP

Components of the sea urchin outer egg jelly layer such as speract drastically change second messenger levels and membrane permeability in sperm. Ion channels are deeply involved in the sperm-egg dialogue in sea urchin and other species. Yet, due to the small size of sperm, studies of ion channels and their modulation by second messengers in sperm are scarce. In this report we offer the first direct evidence that cation-selective channels upwardly regulated by cAMP operate in sea urchin sperm. Due to their poor selectivity among monovalent cations, channel activation in seawater could contribute to sperm membrane repolarization during the speract response.     

External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.     

Improved double immunofluorescence for confocal laser scanning microscopy.

Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using the red-emitting Alexa 568, with amplification of the signal by a biotinylated bridging antibody and labeled streptavidin. This was combined with the green-emitting Alexa 488, which yielded brighter images than fluorescein but exhibited comparable photodegradation. With appropriate controls to ensure the absence of crosstalk between fluorescence channels, these dyes permitted unequivocal demonstration of co-localization. This combination of fluorochromes may also offer advantages for users of instruments equipped with alternative laser systems.     

Two-pore potassium channels in the cardiovascular system

Two-pore domain (K_2P) channels emerged about a decade ago and since then have been an expanding area of interest. This is because their biophysical and pharmacological properties make them good candidates to support background potassium currents and membrane potential in many cell types. There is clear evidence for TREK-1 and TASK-1 in the heart and these channels are likely to regulate cardiac action potential duration through their regulation by stretch, polyunsaturated fatty acids, pH, and neurotransmitters. TREK-1 may also have a critical role in mediating the vasodilator response of resistance arteries to polyunsaturated fatty acids, thus contributing to their protective effect on the cardiovascular system. TASK-1, on the other hand, is a strong candidate for a role in hypoxic vasoconstriction of pulmonary arteries. Many other members of the K_2P channel family have been identified in the cardiovascular system, although their functional roles are still to be demonstrated. This review provides an up to date summary of what is known about the involvement of members of the K_2P channel family in cells of the heart and arterial circulation. Our knowledge of their roles will improve with the rapidly increasing interest in them and as new selective pharmacological tools emerge. As their physiological roles emerge, the K_2P family of potassium channels may offer promising therapeutic solutions to target cardiovascular diseases. EBSA satellite meeting: ion channels, Leeds, July 2007.     

A new approach to insect-pest control—combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity

Voltage-sensitive sodium channels are responsible for the generation of electrical signals in most excitable tissues and serve as specific targets for many neurotoxins. At least seven distinct classes of neurotoxins have been designated on the basis of physiological activity and competitive binding studies. Although the characterization of the neurotoxin receptor sites was predominantly performed using vertebrate excitable preparations, insect neuronal membranes were shown to possess similar receptor sites. We have demonstrated that the two mutually competing antiinsect excitatory and depressant scorpion toxins, previously suggested to occupy the same receptor site, bind to two distinct receptors on insect sodium channels. The latter provides a new approach to their combined use in insect control strategy. Although the sodium channel receptor sites are topologically separated, there are strong allosteric interactions among them. We have shown that the lipid-soluble sodium channel activators, veratridine and brevetoxin, reveal divergent allosteric modulation on scorpion α-toxins binding at homologous receptor sites on mammalian and insect sodium channels. The differences suggest a functionally important structural distinction between these channel subtypes. The differential allosteric modulation may provide a new approach to increase selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective toxins. Thus, a comparative study of neurotoxin receptor sites on mammalian and invertebrate sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins, and may offer new targets and approaches to the development of highly selective pesticides.