Physics of Ion Channels

We review the basic physics involved in transport of ions across membrane channels in cells. Electrochemical forces that control the diffusion of ions are discussed both from microscopic and macroscopic perspectives. A case is made for use of Brownian dynamics as the minimal phenomenological model that provides a bridge between experiments and more fundamental theoretical approaches. Application of Brownian and molecular dynamics methods to channels with known molecular structures is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ion Channels and Cancer

Membrane ion channels are essential for cell proliferation and appear to have a role in the development of cancer. This has initially been demonstrated for potassium channels and is meanwhile also suggested for other cation channels and Cl⁻ channels. For some of these channels, like voltage-gated ether à go-go and Ca²⁺-dependent potassium channels as well as calcium and chloride channels, a cell cycle-dependent function has been demonstrated. Along with other membrane conductances, these channels control the membrane voltage and Ca²⁺ signaling in proliferating cells. Homeostatic parameters, such as the intracellular ion concentration, cytosolic pH and cell volume, are also governed by the activity of ion channels. Thus it will be an essential task for future studies to unravel cell cycle-specific effects of ion channels and non-specific homeostatic functions. When studying the role of ion channels in cancer cells, it is indispensable to choose experimental conditions that come close to the in vivo situation. Thus, environmental parameters, such as low oxygen pressure, acidosis and exposure to serum proteins, have to be taken into account. In order to achieve clinical application, more studies on the original cancer tissue are required, and improved animal models. Finally, it will be essential to generate more potent and specific inhibitors of ion channels to overcome the shortcomings of some of the current approaches.     

Voltage-Gated Lipid Ion Channels

Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current histograms in patch-experiments on lipid membranes. We derived a theoretical current-voltage relationship for pores in lipid membranes that describes the experimental data very well when assuming an asymmetric membrane. We determined the equilibrium constant between closed and open state and the open probability as a function of voltage. The voltage-dependence of the lipid pores is found comparable to that of protein channels. Lifetime distributions of open and closed events indicate that the channel open distribution does not follow exponential statistics but rather power law behavior for long open times.     

酸敏感离子通道研究进展

组织酸化是生理和病理下常见的现象.神经元可以通过酸敏感的离子通道（ASICs）来感受细胞周围的pH值的降低.ASICs属于NaC/DEG家族的一个成员.目前,已发现了6个ASICs亚基,它们在外周和中枢神经系统中广泛表达,其同聚体和异聚体通道有着各种不同的电生理学特性.ASICs在机体感觉尤其是痛觉中起着至关重要的作用.     

NALCN Ion Channels Have Alternative Selectivity Filters Resembling Calcium Channels or Sodium Channels

NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE) or sodium channels (EKEE or EEKE). NALCN channels with alternative calcium, (EEEE) and sodium, (EKEE or EEKE) -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K)-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG⁺ impermeant and blockable with 10 µM Gd³⁺ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2):371-83.     

GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ～20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between “deep” and “shallow” binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as “area reservoirs” leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.     

Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors

Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such “Cys-less” receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the “Cys-loop” proline is absolutely conserved, suggesting the more fitting name “Pro loop” for that motif, and “Pro-loop receptors” for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.     

Mechanosensation and pain

The ability of cells to detect and transduce mechanical stimuli impinging on them is a fundamental process that underlies normal cell growth, hearing, balance, touch, and pain. Surprisingly, little research has focused on mechanotransduction as it relates to the sensations of somatic touch and pain. In this article we will review data on the wealth of different mechanosensitive sensory neurons that innervate our main somatic sense organ the skin. The role of different types of mechanosensitive sensory neurons in pain under physiological and pathophysiological conditions (allodynia and hyperalgesia) will also be reviewed. Finally, recent work on the cellular and molecular mechanisms by which mechanoreceptive sensory neurons signal both innocuous and noxious sensation is evaluated in the context of pain.     

高等植物液泡离子通道

目前为止,已经发现高等植物液泡膜上存在众多的离子通道,本文主要对这些离子通道的特征、生理功能以及调节的国内外研究现状作一综述。     

锌离子对配体门控型离子通道的调节作用

在中枢神经系统(central nervous system,CNS)中,锌离子对配体门控型离子通道具有重要的调节作用。锌离子随着神经元的活动从突触前膜的囊泡中释放到突触间隙,对突触内受体进行调控。锌离子抑制N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)型谷氨酸受体的活性,而对非NMDA型谷氨酸受体的调控具有多样性。由γ氨基丁酸(γ-aminobutyric acid,GABA)受体所介导的抑制性突触传递活动也受到锌离子的抑制;而锌离子对glycine受体则呈现出浓度依赖的双向调节效应。病理条件下,锌离子参与了兴奋性细胞毒作用所触发的神经元凋亡过程。本文主要阐述了在CNS中,锌离子对配体门控型离子通道所介导的突触传递活动的调控作用,以及这些调控作用的生理功能和病理意义。     

Toward Understanding Protocell Mechanosensation

Mechanosensitive (MS) channels can prevent bacterial bursting during hypo-osmotic shocks by responding to increases in lateral tension at the membrane level through an integrated and coordinated opening mechanism. Mechanical regulation in protocells could have been one of the first mechanisms to evolve in order to preserve their integrity against changing environmental conditions. How has the rich functional diversity found in present cells been created throughout evolution, and what did the primordial MS channels look like? This review has been written with the aim of identifying which factors may have been important for the appearance of the first osmotic valve in a prebiotic context, and what this valve may have been like. It highlights the mechanical properties of lipid bilayers, the association of peptides as aggregates in membranes, and the conservation of sequence motifs as central aspects to understand the evolution of proteins that gate below the tension required for spontaneous pore formation and membrane rupture. The arguments developed here apply to both MscL and MscS homologs, but could be valid to mechano-susceptible proteins in general.     

Gating Transition of Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are an important family of membrane proteins and play key roles in physiological processes, including signal transduction at chemical synapses. Here, we study the conformational changes associated with the opening and closing of the channel pore. Based on recent crystal structures of two prokaryotic members of the family in open and closed states, respectively, mixed elastic network models are constructed for the transmembrane domain. To explore the conformational changes in the gating transition, a coarse-grained transition path is computed that smoothly connects the closed and open conformations of the channel. We find that the conformational transition involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition.     

Measuring Ion Channels on Solid Supported Membranes

Application of solid supported membranes (SSMs) for the functional investigation of ion channels is presented. SSM-based electrophysiology, which has been introduced previously for the investigation of active transport systems, is expanded for the analysis of ion channels. Membranes or liposomes containing ion channels are adsorbed to an SSM and a concentration gradient of a permeant ion is applied. Transient currents representing ion channel transport activity are recorded via capacitive coupling. We demonstrate the application of the technique to liposomes reconstituted with the peptide cation channel gramicidin, vesicles from native tissue containing the nicotinic acetylcholine receptor, and membranes from a recombinant cell line expressing the ionotropic P2X₂ receptor. It is shown that stable ion gradients, both inside as well as outside directed, can be applied and currents are recorded with an excellent signal/noise ratio. For the nicotinic acetylcholine receptor and the P2X₂ receptor excellent assay quality factors of Z′ = 0.55 and Z′ = 0.67, respectively, are obtained. This technique opens up new possibilities in cases where conventional electrophysiology fails like the functional characterization of ion channels from intracellular compartments. It also allows for robust fully automatic assays for drug screening.