Multi-ion conduction bands in a simple model of calcium ion channels

We report self-consistent Brownian dynamics simulations of a simple electrostatic model of the selectivity filters (SF) of calcium ion channels. They reveal regular structure in the conductance and selectivity as functions of the fixed negative charge Qf at the SF. This structure consists of distinct regions of high conductance (conduction bands) M0, M1, M2 separated by regions of zero-conductance (stop-bands). Two of these conduction bands, M1 and M2, demonstrate high calcium selectivity and prominent anomalous mole fraction effects and can be identified with the L-type and RyR calcium channels.Comment: 14 pages, 9 figures, 38 reference

Spatiotemporal expression of TRPM4 in the mouse cochlea

The present study was conducted to elucidate the presence of the transient receptor potential cation channel subfamily M member 4, TRPM4, in the mouse inner ear. TRPM4 immunoreactivity (IR) was found in the cell body of inner hair cells (IHCs) in the organ of Corti in the apical side of marginal cells of the stria vascularis, in the apical portion of the dark cells of the vestibule, and in a subset of the type II neurons in the spiral ganglion. Subsequently, changes in the distribution and expression of TRPM4 in the inner ear during embryonic and postnatal developments were also evaluated. Immunohistochemical localization demonstrated that the emergence of the TRPM4-IR in IHCs occurs shortly before the onset of hearing, whereas that in the marginal cells happens earlier, at the time of birth, coinciding with the onset of endolymph formation. Furthermore, semiquantitative real-time PCR assay showed that expressions of TRPM4 in the organ of Corti and in the stria vascularis increased dramatically at the onset of hearing. Because TRPM4 is a Ca2+-activated monovalent-selective cation channel, these findings imply that TRPM4 contributes to potassium ion transport, essential for the signal transduction in IHCs and the formation of endolymph by marginal cells. © 2014 Wiley Periodicals, Inc

Single mechanosensitive and ca(2+)-sensitive channel currents recorded from mouse and human embryonic stem cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca(2+) permeant cation channels (MscCa) and large conductance (200 pS) Ca(2+)-sensitive K(+) (BK(Ca2+)) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK(Ca2+) channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca(2+)-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca(2+)] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.OH was supported by a travel/stay Grant from Ministerio de Educación y Ciencia (SAB2006-0211) and in the United States by grants from the National Cancer Institute and the Department of Defense. BS and AH are supported by the Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía (PI-0022/ 2008); Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (CTS-6505; INP-2011-1615-900000); FEDER cofunded grants from Instituto de Salud Carlos III (Red TerCel-RD06/0010/0025; PI10/00964), and the Ministry of Health and Consumer Affairs (Advanced Therapies Program TRA-120). CIBERDEM is an initiative of the Instituto de Salud Carlos III.Peer Reviewe

Flufenamic acid as an ion channel modulator

Flufenamic acid has been known since the 1960s to have anti-inflammatory properties attributable to the reduction of prostaglandin synthesis. Thirty years later, flufenamic acid appeared to be an ion channel modulator. Thus, while its use in medicine diminished, its use in ionic channel research expanded. Flufenamic acid commonly not only affects non-selective cation channels and chloride channels, but also modulates potassium, calcium and sodium channels with effective concentrations ranging from 10(-6)M in TRPM4 channel inhibition to 10(-3)M in two-pore outwardly rectifying potassium channel activation. Because flufenamic acid effects develop and reverse rapidly, it is a convenient and widely used tool. However, given the broad spectrum of its targets, experimental results have to be interpreted cautiously. Here we provide an overview of ion channels targeted by flufenamic acid to aid in interpreting its effects at the molecular, cellular, and system levels. If it is used with good practices, flufenamic acid remains a useful tool for ion channel research. Understanding the targets of FFA may help reevaluate its physiological impacts and revive interest in its therapeutic potential. (C) 2013 Elsevier Inc. All rights reserved

Die Bedeutung des TRPM 7-Kanals für die Spontanaktivität der glatten Muskulatur

Ursprung der spontanen phasisch-rhythmischen Kontraktionen glattmuskulärer Organe sind die ICC. Das fundamentale Schrittmacherereignis ist trotz seiner hohen klinischen Relevanz bislang nicht eindeutig identifiziert. Ein neuer Kandidat für den Schrittmacherstrom soll der TRPM7-Kanal sein. Diese Hypothese wird innerhalb dieser Arbeit sowohl an glattmuskulären Gewebepräparaten von Antrum, Colon, Portalvene und Uterus der Ratte als auch an kultivierten gastralen ICC durch pharmakologische Hemmung des TRPM7-Kanals untersucht

Caractérisation des canaux potassiques du tubule contourné proximal et des propriétés régulatrices des canaux chlorure de la membrane basolatérale des cellules intercalaires du tubule connecteur

A 10 pS chloride channel at the basolateral side of connecting duct intercalated cells shares properties with the cloned ClC-K2 channel. Patch-clamp experiments show that its activity and the number of active channels increase with (i) membrane depolarization (ii) external calcium concentration and (iii) external and internal alkalinization. External alkalinization also shifts the voltage-dependence curve towards negative voltages while internal alkalinization flattens the voltage-dependence curve thereby raising channel activity at negative potentials. These data suggest that extracellular calcium and both extra and intracellular protons modulate ClC-K2 channels activity through an action on the common gate rather than on the protopores present in others ClC channels.The role and the molecular identity of basolateral potassium channels of the proximal convoluted tubule (PCT) are not very well known. RT-PCR results revealed the presence of mRNA encoding the Kir4.2 and Kir5.1 potassium channels subunits in mouse PCT tubular cells, and western blot and immunohistochemistry experiments showed that both proteins are expressed at the basolateral membrane of these cells. The most frequent channel observed by patch-clamp on the basolateral membrane of PCT presents a conductance of 47 pS, an inward rectification induced by intracellular Mg2+, an inhibition by extracellular Ba2+ and an activity dependent on intracellular pH. These electrophysiological properties are consistent with the presence of heteromeric Kir4.2/Kir5.1 channels in the basolateral membrane of mouse PCT. The study of mice knocked out for the Kir4.2-encoding gene Kcnj15 did not highlight a renal phenotype.Un canal chlorure de 10 pS de la membrane basolatérale des cellules intercalaires du canal connecteur présente des propriétés proches de celles de ClC-K2. Nos données de patch clamp montrent que son activité et le nombre de canaux actifs (N) augmentent avec (i) la dépolarisation membranaire (ii) la concentration en calcium externe et (iii) l’alcalinisation extra et intracellulaire. L’alcalinisation extracellulaire déplace la dépendance au voltage vers des potentiels négatifs, alors que l’alcalinisation intracellulaire augmente leur activité à des potentiels négatifs. Ces données suggèrent que le Ca2+ extracellulaire et le pH modulent l’activité des canaux ClC-K2 via une action sur la porte commune plutôt que sur les protopores des autres canaux ClC. Nous avons cherché à établir le rôle et l’identité moléculaire des canaux potassiques basolatéraux du tubule contourné proximal (PCT), peu connus. Nos résultats montrent la présence d’ARNm codant pour les sous-unités Kir4.2 et Kir5.1 dans le PCT et, par western blot et par immunohistochimie, leur expression sur la membrane basolatérale du PCT. Un canal potassique, étudié par patch-clamp, présente des propriétés semblables à celles des canaux hétérotétramériques Kir4.2/Kir5.1 sur la membrane basolatérale du PCT de souris : il présente une conductance de 47 pS, une rectification entrante induite par le Mg2+ intracellulaire, une dépendance de son activité au pHi et un blocage par le Ba2+ extracellulaire. Cependant, un phénotype rénal chez des souris invalidées pour le gène Kcnj15 codant pour Kir4.2 n’a pas pu être mis en évidence

The modulation of Transient Receptor Potential A1 channel by natural and novel semi-synthetic compounds via non-covalent modification

Transient Receptor Potential A1 (TRPA1) is commonly known as the detector of a broad range of noxious chemical agents both exogenous and endogenous. TRPA1 detects these chemicals through a reversible covalent modification mechanism that allows most electrophilic compounds to activate the channel; hence one of the channel’s key roles is to protect the respiratory system from harmful irritants by activating the cough reflex. It has been proposed that TRPA1 is involved in chronic inflammatory diseases of the respiratory system and has been highlighted as a potential drug target for this as well as general pain and inflammation.TRPA1 is also activated by non-covalent mechanisms, which are less well understood. I therefore aimed to gain a further understanding of non-covalent mechanisms of TRPA1 modulation via structure-activity relationship studies using several groups of diverse compounds based on existing TRPA1 agonists.The results reported have shown that compounds based on thymol, carvacrol and fenamic acid have a diverse effect on TRPA1 dependent on small alterations in structure. This highlights the delicate nature of the TRPA1 non-covalent binding sites. The derivatives tested all share one common structural feature; they all have two phenyl rings which are linked via different functional groups and different lengths. It was found that the length of the linker had an effect on the potency of the modulation of TRPA1. In addition to these results NDGA and its semi-synthetic derivative M4N were potent TRPA1 agonists, yet unlike other similar compounds do not desensitise TRPA1, possibly due to their folded structure. Throughout the results, the importance of hydrogen bonding was shown with different functional groups capable of acting as donors or acceptors.Overall the results reported expand the group of non-covalent modulators of TRPA1 and indicated important structural features that must be considered in any future TRPA1 drug development projects