Discovery of diphenyl amine based sodium channel blockers, effective against hNav1.2

The development of new therapies for chronic pain is an area of unmet medical need. Central to pathways of chronic pain is the upregulation of voltage-gated sodium channels. The use of tricyclic antidepressants, which also have sodium channel activity, in chronic pain therapy prompted us to develop novel compounds from this scaffold. Herein, we show that the tricyclic moiety is not needed for effective inhibition of the [³H]-BTX binding site and sodium currents of hNa_v1.2. Our lead compound 6, containing a diphenyl amine motif, demonstrated a 53% inhibitory block of Na_v1.2 currents at 10 μM, which is greater than 50% increase in current block in comparison to the amitriptyline standard. Altogether our study establishes that the tricyclic motif is unnecessary for hNa_v1.2 activity and modification of the amine portion is detrimental to sodium channel block.     

Signalling Via the G Protein-Activated K Channels

The inwardly rectifying K⁺ channels of the GIRK (K_ir3) family, members of the superfamily of inwardly rectifying K⁺ channels (K_ir), are important physiological tools to regulate excitability in heart and brain by neurotransmitters, and the only ion channels conclusively shown to be activated by a direct interaction with heterotrimeric G protein subunits. During the last decade, especially since their cloning in 1993, remarkable progress has been made in understanding the structure, mechanisms of gating, activation by G proteins, and modulation of these channels. However, much of the molecular details of structure and of gating by G protein subunits and other factors, mechanisms of modulation and desensitization, and determinants of specificity of coupling to G proteins, remain unknown. This review summarizes both the recent advances and the unresolved questions now on the agenda in GIRK studies.     

Proteins Associated with α1-Subunit-Containing GABAA Receptors from Bovine Brain

Abstract: In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABA_A receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABA_A receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABA_A receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the α1-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with α1-subunit-containing GABA_A receptors. Results of a biochemical characterization of these G ABA_A receptor- t ubulin complex- a ssociated p roteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.     

Cav1 L-type Ca2+ channel signaling complexes in neurons

Ca_v1 L-type Ca²⁺ channels play crucial and diverse roles in the nervous system. The pre- and post-synaptic functions of Ca_v1 channels not only depend on their intrinsic biophysical properties but also their dynamic regulation by a host of cellular influences. These include protein kinases and phosphatases, G-protein coupled receptors, scaffolding proteins, and Ca²⁺-binding proteins. The cytoplasmic domains of the main pore forming α₁ subunit of Ca_v1 offer a number of binding sites for these modulators, permitting fast and localized regulation of Ca²⁺ entry. Through effects on Ca_v1 gating, localization, and coupling to effectors, protein modulators are efficiently positioned to adjust Ca_v1 Ca²⁺ signals that control neuronal excitability, synaptic plasticity, and gene expression.     

Dynamic properties of stretch-activated K+ channels in adult rat atrial myocytes

The effect of mechanical stress on the heart's electrical activity has been termed mechanoelectric feedback. The response to stretch depends upon the magnitude and the waveform of the stimulus, and upon the timing relative to the cardiac cycle. Stretch-activated ion channels (SACs) have been regarded as the most likely candidates for serving as the primary transducers of mechanical stress. We explored the steady state and dynamic responses of single channels in adult rat atrial cells using the patch clamp with a pressure clamp. Surprisingly, we only observed K⁺-selective SACs, probably of the 2P domain family. The channels were weakly outward rectifying with flickery bursts. In cell attached mode, the mean conductance was 74±14 and 65±16 pS for +60 and −60 mV, respectively (140 mM [K⁺]_out, 2 mM [Mg²⁺]_out and 0 mM [Ca²⁺]_out). The latency of the response to pressure steps was 50–100 ms and the time to peak 400 ms. About half of the channels in cell-attached patches showed adaptation/inactivation where channel activity declined to a plateau of 20–30% of peak in 1 s. The time dependent behavior of these SACs is generally consistent with whole-cell currents observed in chick and rat ventricular cells, although the net current was outward rather than inward.     

基于机械敏感离子通道的超声神经调制研究进展

机械敏感离子通道（mechanosensitive channels,MSCs）是一类分布于各种细胞膜上可将细胞受到的机械刺激转化为电信号或化学信号的特殊膜蛋白。由于机械敏感通道所具有的特性,使其成为超声调控的重要潜在靶点。超声由于具有良好的空间分辨率和聚焦效果,并且理论上可实现无创条件下的全脑范围定位,具有用于进行物理性神经调制和治疗神经系统疾病的潜力。近年来,越来越多的离子通道被鉴定出具有机械敏感特性,但其中有明确报道可以被超声激活的依然数量较少。此外,现阶段超声激励下机械敏感通道的开放过程和机制仍未被阐明。本文着重介绍了大电导机械敏感通道、瞬时受体电位通道、退化蛋白/上皮钠通道、双孔钾通道和Piezo通道等机械敏感离子通道在超声神经调制中的研究进展及其应用,为未来超声神经调制的深入研究和临床应用提供参考。     

OSCA/TMEM63家族离子通道的研究进展

高渗促钙内流蛋白(hyperosmolality-induced [Ca2+]iincrease,OSCA)/跨膜蛋白63 (transmembrane protein 63,TMEM63)家族蛋白是一类多次跨膜蛋白质,它们在真核细胞中有广泛分布.研究表明拟南芥中OSCA1.1蛋白介导了高渗刺激的钙离子内流.进一步研究发现OSCA1.1及其同源蛋白质是机械力敏感的离子通道.高分辨率冷冻电镜结构显示OSCA蛋白是对称的二聚体,每个亚基含有一个离子可通透的孔道.本文将从OSCA通道的功能、结构以及结构与功能的关系几方面介绍该领域的研究进展.     

Epidermal Growth Factor Promotes Uncoupling from Adenylyl Cyclase of the Rat D2S Receptor Expressed in GH4C1 Cells

Abstract: In anterior pituitary cells or when transfected into host cell lines, the D₂ dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D₂ receptors, responds to epidermal growth factor (EGF) by expressing a D₂ receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in α subunit of the G protein G₁₃. In this study we have investigated the effects of EGF on the transduction mechanisms of D₂ receptors in GH4C1 cells transfected and permanently overexpressing the rat short D₂ receptor. Activation of D₂ receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D₂ receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of G₁₃ protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.     

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.     

植物细胞质膜离子通道研究进展

多种有机和无机离子作为重要的营养物质、渗透物质、辅酶和信号分子, 参与植物生殖、生长发育和逆境反应等多种生物学过程。离子通道是离子跨质膜和内膜运动的重要渠道和动态调控因子, 直接影响和调控细胞内离子浓度及亚细胞分布的动态变化。目前, 植物尤其是模式植物拟南芥(Arabidopsis thaliana)的多个离子通道家族被先后鉴定出来, 其中部分离子通道蛋白定位在细胞质膜上, 其基本生物学功能, 诸如蛋白结构、离子选择性和通透性、门控特点、活性调控机理以及不同离子通道之间的协同关系等均取得重要进展。该文概要介绍近年来植物细胞质膜离子通道方面的研究进展。     

Calcium channel γ subunits provide insights into the evolution of this gene family

The γ subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct γ subunits have been described from human and/or mouse. The first identified member of this group of proteins, γ₁, is a component of the L-type calcium channel expressed in skeletal muscle. A second member, γ₂, identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel γ subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian γ subunits suggests the following relationship ((((γ₂, γ₃), (γ₄, γ₈)), (γ₅, γ₇)), (γ₁, γ₆)) that indicates that they evolved from a common ancestral γ subunit via gene duplication. Our analysis reveals that the novel γ subunit γ₆ most closely resembles γ₁ and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other γ subunits. Rat γ subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of γ₁ mRNA and the long isoform of γ₆ mRNA is most robust in skeletal muscle, while γ₆ is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the γ subunits, we propose that γ₁ and γ₆ may share common physiological functions distinct from the other homologous γ subunits.     

A functional NMR for membrane proteins: dynamics,ligand binding,and allosteric modulation

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand‐induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.     

Role of K(ATP) channels in protection against neuronal excitatory insults

ATP-sensitive K⁺ (K_ATP) channels that are gated by intracellular ATP/ADP concentrations are a unique subtype of potassium channels and play an essential role in coupling intracellular metabolic events to electrical activity. Opening of K_ATP channels during energy deficits in the CNS induces efflux of potassium ions and in turn hyperpolarizes neurons. Thus, activation of K_ATP channels is thought to be able to counteract excitatory insults and protect against neuronal death. In this review, we bring together recent studies about what kinds of molecules are needed to build and regulate arrays of K_ATP channel functions in the CNS neurons. We propose a model to explain how K_ATP channel activation regulates glutamate release from the pre-synaptic terminals and how this regulation protects against ischemic neuronal injury and epilepsy.     

Recent advances in ion channel research

The field of ion channels has entered into a rapid phase of development in the last few years, partly due to the breakthroughs in determination of the crystal structures of membrane proteins and advances in computer simulations of biomolecules. These advances have finally enabled the long-dreamed goal of relating function of a channel to its underlying molecular structure. Here we present simplified accounts of the competing permeation theories and then discuss their application to the potassium, gramicidin A and calcium channels.     

Effects of hypochlorite disinfection on the response of barley aleurone layers to gibberellic acid

Although the use of hypochlorite to disinfect seeds is widespread, the effects on tissues isolated from them have been largely ignored. Disinfection of barley ( Hordeum vulgare L. cv. Himalaya) half-seeds with hypochlorite solutions of ≥1.0% (w/v) available chlorine caused the pericarp to separate from the underlying tissues. Aleurone layers isolated from these grains had lower rates of oxygen consumption and released significantly less protein, PO₄³⁻ Mg²⁺, K⁺ and amylase (EC 3.2.1.1) into the medium in response to gibberellic acid (GA₃) than layers isolated from grains disinfected with a 0.1% hypochlorite solution. Disinfection with 1.0% hypochlorite also quantitatively altered the spectrum of proteins released into the incubation medium by the layers in response to GA₃.     

Late sodium current in failing heart: Friend or foe?

Most cardiac Na⁺ channels open transiently upon membrane depolarization and then are quickly inactivated. However, some channels remain active, carrying the so-called persistent or late Na⁺ current (I_NaL) throughout the action potential (AP) plateau. Experimental data and the results of numerical modeling accumulated over the past decade show the emerging importance of this late current component for the function of both normal and failing myocardium. I_NaL is produced by special gating modes of the cardiac-specific Na⁺ channel isoform. Heart failure (HF) slows channel gating and increases I_NaL, but HF-specific Na⁺ channel isoform underlying these changes has not been found. Na⁺ channels represent a multi-protein complex and its activity is determined not only by the pore-forming subunit but also by its auxiliary β subunits, cytoskeleton, calmodulin, regulatory kinases and phosphatases, and trafficking proteins. Disruption of the integrity of this protein complex may lead to alterations of I_NaL in pathological conditions. Increased I_NaL and the corresponding Na⁺ flux in failing myocardium contribute to abnormal repolarization and an increased cell Ca²⁺ load. Interventions designed to correct I_NaL rescue normal repolarization and improve Ca²⁺ handling and contractility of the failing cardiomyocytes. This review considers (1) quantitative integration of I_NaL into the established electrophysiological and Ca²⁺ regulatory mechanisms in normal and failing cardiomyocytes and (2) a new therapeutic strategy utilizing a selective inhibition of I_NaL to target both arrhythmias and impaired contractility in HF.     

Antibodies Against Rat Brain Vesicle-Associated Membrane Protein (Synaptobrevin) Prevent Inhibition of Acetylcholine Release by Tetanus Toxin or Botulinum Neurotoxin Type B

Abstract: The Shaw-type K⁺ channel K_v3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K⁺ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to K_v 3.1 channels expressed transiently in Xenopus oocytes. K_v 3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13-dibutyrate decreased K_v 3.1 currents. Based on these results, we find that the stable expression of voltage-gated K⁺ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.     

Lysophosphatidic Acid Sensitises Ca Influx through Mechanosensitive Ion Channels in Cultured Lens Epithelial Cells

We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca²⁺]_i in the presence of LPA and this increase was concentration-dependent (1–10 μM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca²⁺]_i. The mechanical stress-induced increase in [Ca²⁺]_i in the presence of LPA was inhibited by removing extracellular Ca²⁺ or by addition of Gd³⁺, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca²⁺ influx through cation-selective mechanosensitive channels, but does not sensitise Ca²⁺ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca²⁺ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca²⁺ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca²⁺]_i induced by mechanical stress.