Transient receptor potential ion channels and animal sensation: lessons from Drosophila functional research

Ion channels of the transient receptor potential (TRP) superfamily are non-selective cationic channels with six transmembrane domains. The TRP channel made its first debut as a light-gated Ca2+ channel in Drosophila. Recently, research on animal sensation in Drosophila disclosed other members of the TRP family that are required for touch sensation and hearing as well as the sensation of painful stimuli.     

Structural biology of thermoTRPV channels

Essential for physiology, transient receptor potential (TRP) channels constitute a large and diverse family of cation channels functioning as cellular sensors responding to a vast array of physical and chemical stimuli. Detailed understanding of the inner workings of TRP channels has been hampered by a lack of atomic structures, though structural biology of TRP channels has been an enthusiastic endeavor since their molecular identification two decades ago. These multi-domain integral membrane proteins, exhibiting complex polymodal gating behavior, have been a challenge for traditional X-ray crystallography, which requires formation of well-ordered protein crystals. X-ray structures remain limited to a few TRP channel proteins to date. Fortunately, recent breakthroughs in single-particle cryo-electron microscopy (cryo-EM) have enabled rapid growth of the number of TRP channel structures, providing tremendous insights into channel gating and regulation mechanisms and serving as foundations for further mechanistic investigations. This brief review focuses on recent exciting developments in structural biology of a subset of TRP channels, the calcium-permeable, non-selective and thermosensitive vanilloid subfamily of TRP channels (TRPV1-4), and the permeation and gating mechanisms revealed by structures.     

昆虫分子生物学的一些进展：神经递质和离子通道

昆虫分子生物学的一些进展：神经递质和离子通道翟启慧（中国科学院动物研究所北京１０００８０）１神经递质神经递质（ｎｅｕｒｏｔｒａｎ。ｍｉｔｔｅｒ）是在化学突触神经ｆＧ。１问传递信息的化学物质。神经递质有许多不同类型,如乙酸胆碱、丫一氨基丁酸、生物胺等。...     

High‐resolution structures of transient receptor potential vanilloid channels: Unveiling a functionally diverse group of ion channels

Transient receptor potential vanilloid (TRPV) channels are part of the superfamily of TRP ion channels and play important roles in widespread physiological processes including both neuronal and non‐neuronal pathways. Various diseases such as skeletal abnormalities, chronic pain, and cancer are associated with dysfunction of a TRPV channel. In order to obtain full understanding of disease pathogenesis and create opportunities for therapeutic intervention, it is essential to unravel how these channels function at a molecular level. In the past decade, incredible progress has been made in biochemical sample preparation of large membrane proteins and structural biology techniques, including cryo‐electron microscopy. This has resulted in high resolution structures of all TRPV channels, which has provided novel insights into the molecular mechanisms of channel gating and regulation that will be summarized in this review.     

Functional characteristic and molecular topology of voltage independent sodium channels

The channel proteins so far known are transmembrane oligomers arranged in a manner that the polar residues are lining the central ion-conducting hydrophilic pore. In the last decade, electrophysiology and molecular biology studies revealed the principal similarity in the functional properties and membrane topology within a large family of sodium-conducting channels. Amiloride-sensitive channels are expressed in the apical membranes of renal epithelia. Moreover, in different mammalian cells non-voltage-gated sodium-selective channels have been recently found. According to molecular cloning of the respective DNAs and amino acid sequence analysis, epithelial channel subunits, degenerins and some other channel proteins display a significant homology in the regions forming two presumable transmembrane domains. This paper reviews some relevant data and current opinions of the superfamily of sodium-conducting cation channels.     

背根神经节神经元阿片受体和离子通道的研究进展

阿片及阿片受体与外周神经系统镇痛机制的研究,随着分子生物学技术的发展,已在受体的分子结构、形态学、分子药理学、离子通道和细胞内信号转导系统等方面取得了显著进展。μ、δ、κ阿片受体分子结构上的部分差异决定了它们各自的功能特征。三种受体在初级感觉神经元分布的比例不同,但都能介导细胞Ｃａ＾２＋通道的抑制和Ｋ＾＋电流增加及减少。阿片受体和通道之间由多种第二信使系统偶联。分子药理学研究表明它们还存在亚型受体     

Lights,cytoskeleton, action: Optogenetic control of cell dynamics

Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein–protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.     

Use of toxins to study potassium channels

Potassium channels comprise groups of diverse proteins which can be distinguished according to each member's biophysical properties. Some types of K⁺ channels are blocked with high affinity by specific peptidyl toxins. Three toxins, charybdotoxin, iberiotoxin, and noxiustoxin, which display a high degree of homology in their primary amino acid sequences, have been purified to homogeneity from scorpion venom. While charybdotoxin and noxiustoxin are known to inhibit more than one class of channel (i.e., several Ca²⁺-activated and voltage-dependent K⁺ channels), iberiotoxin appears to be a selective blocker of the high-conductance, Ca²⁺-activated K⁺ channel that is present in muscle and neuroendocrine tissue. A distinct class of small-conductance Ca²⁺-activated K⁺ channel is blocked by two other toxins, apamin and leiurotoxin-1, that share no sequence homology with each other. A family of homologous toxins, the dendrotoxins, have been purified from venom of various related species of snakes. These toxins inhibit several inactivating voltage-dependent K⁺ channels. Although molecular biology approaches have been employed to identify and characterize several species of voltagegated K⁺ channels, toxins directed against a particular channel can still be useful in defining the physiological role of that channel in a particular tissue. In addition, for those K⁺ channels which are not yet successfully probed by molecular biology techniques, toxins can be used as biochemical tools with which to purify the target protein of interest.     

Toward an understanding of structure and function of ion channels

The second half of the 1980s is certain to be considered a turning point in the study of ion channels. Within the last few years, monumental advances in the application of molecular biology, single-channel recording, and direct molecular characterization have been brought to bear on the problem of relating the molecular structure of the ion channel proteins to their function in the cell membrane. Structure-function relationships can now be studied at a level of detail that was unimagined a decade ago. Recently, advances made with the techniques of molecular biology appear to have dominated the literature in this field; however, innovative strategies of structural characterization and electrical measurements of functioning channels in native and artificial membranes continue to break new ground. This paper is a selective review of current progress in understanding structure-function relationships in ion channels. The relative usefulness of determining amino acid sequences of channel proteins together with the resulting deductions about 3-dimensional structure and function will be evaluated with respect to the potential importance of studying the channel molecules more directly by biochemical, immunological, and electrophysiological methods. A full understanding of the details of channel structure and its relationship to function may be realized in the near future as a result of the interdisciplinary application of biophysical, biochemical, and molecular biological techniques.     

Fast,repetitive light-activation of CaV3.2 using Channelrhodopsin 2

Channelrhodopsin-2 (ChR2) is a light-gated ion channel that is successfully used in neurosciences to depolarize cells with blue light. In this regard control of membrane voltage with light opens new perspectives for the characterization of ion channels and the search for inhibitors or modulators. Here, we report a control of membrane potential with ChR2 and the potassium channel mTrek for the purpose of screening for ion channel specific drugs. To verify principle we have chosen the voltage gated calcium channel Ca_V3.2 as potential drug target. For this purpose we transfected the ChR2 gene into a HEK293T-cell line that permanently expresses Ca_V3.2 and the K-channel mTrek. The resting potential was adjusted with low concentration of extracellular potassium ions whereas transient depolarization was achieved by activation of ChR2 with short pulses of blue light. Calcium ion influx through Ca_V3.2 was monitored by observing fura-2 fluorescence. This approach allowed a repetitive activation of Ca_V3.2. The Ca²⁺ influx was specifically blocked by the inhibitor mibefradil. Since this assay is genetically-encoded, it may be employed for a variety of voltage-gated calcium channels and should be applicable to multi-well reader formats for high-throughput screening.     

心肌细胞钾通道的研究进展

从分子水平上看,所有钾通道都是由一个基因家族中的基因所编码的４个亚基组成,通道的失活门控机制在Ｎ型、Ｃ型和Ｐ型三种,通道的孔道结构均在跨膜片段Ｓ５至Ｓ６之间,尽管各种钾通道在分子结构上的共处远多于不同之处,但每种钾通道的个性表现却有非常重要的生理意义,迄今为止在心肌细胞上发现的８种钾通道的电导值,门控动力学特征、离子动力学特征,通道激动剂,阻断剂和调制剂均不同。     

Modeling of the pore domain of the GLUR1 channel: homology with K+ channel and binding of channel blockers

Molecular models of the M2 segments of the GluR1 channel have been elaborated using a molecular mechanics approach. The models are based on the homology between pore-lining segments of AMPA receptor channels and the KcsA K+ channel and on cyclic H bonds at the Q/R site of the AMPA receptor channel. The N-terminal region of an M2 segment of the channel is assumed, like that of the K+ channel, to adopt a helical conformation. Due to a deletion, the C-terminal end of the M2 segment of the AMPA receptor is more stretched than that of the K+ channel. As a result, only a single oxygen ring may be exposed to the AMPA receptor channel pore. Data on the block of AMPA receptor channels by dicationic adamantane derivatives have been used to select the most relevant model. The model with the oxygen of a Gly residue (position +2 from the Q/R site) exposed to the pore best fits the experimental data. This model also fits experimental data for another class of AMPA receptor antagonists, the polyamine amides. According to the model, the side-chains of the C-terminal residues are involved in intra-receptor interactions that stabilize the structure of the channel rather than in interactions with ions in the pore.     

植物质膜钾离子转运体研究进展

近年,随着分子生物学技术的不断发展和广泛应用,有关植物质膜钾离子转运体的研究取得重要进展。目前已经克隆到多种质膜钾离子转运体基因并对钾离子转运体生化特性以及结构功能进行了广泛研究。研究认为,质膜钾离子转运体可分为钾离子载体和钾离子通道。钾离子通道又可分为内向性K+通道α亚基、K+通道β亚基及外向性K+通道等三类。本文对上述质膜钾离子转运体的生化特性以及结构功能研究的进展进行了综述。     

Pump-like channelrhodopsins: Not just bridging the gap between ion pumps and ion channels

Channelrhodopsins are microbial rhodopsins that work as light-gated ion channels. Their importance has become increasingly recognized due to their ability to control the membrane potential of specific cells in a light-dependent manner. This technology, termed optogenetics, has revolutionized neuroscience, and numerous channelrhodopsin variants have been isolated or engineered to expand the utility of optogenetics. Pump-like channelrhodopsins (PLCRs), one of the recently discovered channelrhodopsin subfamilies, have attracted broad attention due to their high sequence similarity to ion-pumping rhodopsins and their distinct properties, such as high light sensitivity and ion selectivity. In this review, we summarize the current understanding of the structure-function relationships of PLCRs and discuss the challenges and opportunities of channelrhodopsin research.     

Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function: II. RELATIONSHIP OF THE PHOTOCHEMICAL REACTION CYCLE TO CHANNEL CURRENTS*

Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli.     

Structure of glutamate receptor ion channels and mechanisms of their blockade by organic cations

Structural determinants of blocking the glutamate receptors of AMPA and NMDA subtypes, were studied. Close location of hydrophobic and ammonium groups is necessary for affective blocking of the NMDA receptor channels, whereas blockers of the AMPA receptor channels have a distance of about 10 angstroms between these two groups. Models of the channels meeting these topographic data have been devised using a molecular mechanics approach. The accomplished studies revealed molecular basis of channel blockade of the NMDA and AMPA receptors. This may allow designing predictable new blocking compounds with a desired selectivity.     

First images of a glutamate receptor ion channel: oligomeric state and molecular dimensions of GluRB homomers.

We have expressed, purified, and characterized glutamate receptor ion channels (GluR) assembled as homomers of the subunit GluRB. For the first time, single-milligram quantities of biochemically homogeneous GluR have been obtained. The protein exhibits the expected pharmacological profile and a high specific activity for ligand binding. Density-gradient centrifugation reveals a uniform oligomeric assembly and a molecular mass suggesting that the channel is a tetramer. On the basis of electron microscopic images, the receptor appears to form an elongated structure that is visualized in several orientations. The molecular dimensions of the molecule are approximately 11 x 14 x 17 nm, and solvent-accessible features can be seen; these may contribute to formation of the ion-conducting pathway of the channel. The channel dimensions are consistent with an overall 2-fold symmetric assembly, suggesting that the tetrameric receptor may be a dimer of dimers.