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18. Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms.

Author

Wray, Robin, Iscla, Irene, Kovacs, Zoltan, Junmei Wang, and Blount, Paul

Abstract

The bacterial mechanosensitive channel of large conductance (MscL) normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. Opening the large pore of MscL inappropriately is detrimental to the cell, and thus it has been speculated to be a potential antibiotic target. Although MscL is one of the best studied mechanosensitive channels, no chemical that influenced bacterial growth by modulating MscL is known. We therefore used a high-throughput screen to identify compounds that slowed growth in an MscL-dependent manner. We characterized 2 novel sulfonamide compounds identified in the screen. We demonstrated that, although both increase MscL gating, one of these compounds does not work through the folate pathway, as other antimicrobial sulfonamides; indeed, the sulfonamide portion of the compound is not needed for activity. The only mode of action appears to be MscL activation. The binding pocket is where an α-helix runs along the cytoplasmic membrane and interacts with a neighboring subunit; analogous motifs have been observed in several prokaryotic and eukaryotic channels. The data not only demonstrate that MscL is a viable antibiotic target, but also give insight into the gating mechanisms of MscL, and they may have implications for developing agonists for other channels. [ABSTRACT FROM AUTHOR]

Published
2019
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21. A new antibiotic with potent activity targets MscL

Author

Iscla, Irene, primary, Wray, Robin, additional, Blount, Paul, additional, Larkins-Ford, Jonah, additional, Conery, Annie L, additional, Ausubel, Frederick M, additional, Ramu, Soumya, additional, Kavanagh, Angela, additional, Huang, Johnny X, additional, Blaskovich, Mark A, additional, Cooper, Matthew A, additional, Obregon-Henao, Andres, additional, Orme, Ian, additional, Tjandra, Edwin S, additional, Stroeher, Uwe H, additional, Brown, Melissa H, additional, Macardle, Cindy, additional, van Holst, Nick, additional, Ling Tong, Chee, additional, Slattery, Ashley D, additional, Gibson, Christopher T, additional, Raston, Colin L, additional, and Boulos, Ramiz A, additional

Published
2015
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24. Serotonergic modulatio of a sensory-motor circuit in the leech hirudo medicinalis

Author

Iscla, Irene and Szczupak, Lidia

Subjects
NEUROMODULATION, SENSORY-MOTOR CIRCUIT, SANGUIJUELA HIRUDO MEDICINALIS, SEROTONIN, NEUROMODULACION, CIRCUITO SENSORO-MOTOR, LEECH HIRUDO MEDICINALIS, SEROTONINA
Abstract

El rol neuromodulador de la serotonina ha sido conservado a lo largo de laevolución y se ha observado en invertebrados y en vertebrados. Los invertebradoshan sido utilizados como modelos de estudio de la modulación por serotonina, yaque la simpleza de sus sistemas nerviosos, permite correlacionar alteracionescomportamentales con modificaciones en los circuitos neuronales subyacentes adichos comportamientos. La posibilidad de identificar inequívocamente a lasneuronas serotoninérgicas, permite correlacionar su actividad con los efectosatribuidos a la serotonina. La serotonina es un importante neuromodulador en la sanguijuela: intervieneen la modulación de varios comportamientos tales como la alimentación, elacortamiento defensivo y la natación. Las neuronas serotoninérgicas Retziusconstituyen su fuente principal de liberación. La estimulación mecánica de la pieldel animal produce la activación de las células mecanosensoriales P. Dichaactivación es suficiente para evocar diversas respuestas motoras y a su vez, enparalelo, activar a las neuronas Retzius. Este trabajo de tesis tuvo como objetivo elestudio de la modulación serotoninérgica de un circuito sensoro-motor en el sistemanervioso central de la sanguijuela Hirudo medicinalis, basándose en la hipótesis deque la salida motora final estará determinada por la interacción de la via sensoromotoray la vía sensoro-moduladora. En la primera parte del trabajo se caracterizó la vía sensoro-motora entre lasneuronas mecanosensoriales sensibles a presión (P) y las motoneuronas annuluserectus (AE), que inervan los músculos responsables de las erección de los ánulos enque se divide la pared corporal del animal. Losresultados muestran que: - La motoneurona AE recibe dos señales simultáneas ante la activación de laneurona mecanosensorial P: una excitatoria y otra inhibitoria. La fase excitatoriaestá mediada por una sinápsis directa y la inhibitoria por una interacciónpolisináptica. - La interneurona pasiva 151 ejerce un rol modulador sobre la sinápsis P-AE,interactuando con la motoneurona AE y con la(s) interneurona(s) que media(n) lafase inhibitoria de la conexión P-AE. En la segunda parte del trabajo se analizó el efecto de la serotonina exógenay de antagonistas serotoninérgicos sobre las propiedades intrínsecas de las neuronas P y AEy sobre la interacción P-AE. - La aplicación de serotonina exógena produjo la hiperpolarización de las neuronas AE, por activación de una conductancia a cloro sensible a los antagonistasmetiotepina y mianserina. - La serotonina exógena causó una disminución de la amplitud de la faseinhibitoria de la interacción P-AE,y los antagonistas metiotepina y mianserinabloquearon este efecto. - La serotonina endógena ejercería una inhibición sobre la interacción P-AE. The role of serotonin as neuromodulator has been conserved along evolution,being observed in vertebrates as well as in invertebrates. Invertebrates have beenextensively used as models to study serotonin modulation because their simplenervous systems allow correlating behavioral changes with changes in the pattern ofactivity of the neural circuits that underlie specific behaviors. The preciseidentification of serotonin-containing neurons allows correlating their activity withthe effects attributed to this amine. Serotonin is an important neuromodulator in the leech: it is responsible forthe expression of feeding behavior and modulates shortening and swimming. Retziusneurons contain approximately half of the total ganglionic serotonin. Mechanicalstimulation of the skin activates mechanosensory cells sensitive to pressure, P cells. This activation is capable to evoke several motor responses and in parallel toactivate the serotonin-containing Retzius cells. The goal of this thesis work has beento evaluate the serotonin modulation of a sensory-motor circuit in the centralnervous system of the leech Hirudo medicinalis, based on the hypothesis that thefinal motor output results of the interaction between a sensory-modulatory and asensory-motor pathways. The aim of the first part of this work was to characterize the interactionbetween a pair of specific sensory and motor neurons: the P mechanosensoryneurons and the AE(annulus erector) cells, a pair of motor neurons that innervatethe muscles responsible for erecting the annuli into sharp ridges. The results showthat - The response of the AE neurons was composed of excitatory postsynapticpotentials (EPSPs) and of inhibitory postsynaptic potentials (IPSPs). The EPSPswere transmitted via a monosynaptic pathway, and the IPSPs via a polysynapticone. - The non-spiking neuron 151 is able to modulate the P-AE interaction, by meansof rectifying electrical synapses with AE motoneuron and with the interneuronallayer that mediates the inhibitory response. In the second part of this work we studied the effects of serotonin andserotonin receptor antagonists on the P-AE interaction and on the intrinsicproperties of the neurons involved. - Exogenously applied serotonin produced the hyperpolarization of AEmotorneurons, due to the activation of a chloride conductance that was sensitiveto methiothepine and mianserin. - Exogenously applied serotonin specifically diminished the amplitude of theinhibitory response to P cell stimulation, and this effect was also sensitive tomethiothepine and mianserin. - Endogenous serotonin would exert an inhbitory modulation on this sensory-motorcircuit. Fil: Iscla, Irene. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Published
2001

27. A competing hydrophobic tug on L596 to the membrane core unlatches S4-S5 linker elbow from TRP helix and allows TRPV4 channel to open.

Author

Jinfeng Teng, Loukin, Stephen H., Anishkin, Andriy, Ching Kung, Booth, Ian R., Iscla, Irene, and Martinac, Boris

Subjects
TRP channels, LIPIDS, AMINO acids, XENOPUS, TRPV cation channels, GAIN-of-function mutations
Abstract

We have some generalized physical understanding of how ion channels interact with surrounding lipids but few detailed descriptions on how interactions of particular amino acids with contacting lipids may regulate gating. Here we discovered a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (transient receptor potential vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between the indole of W733 of the TRP helix and the backbone oxygen of L596 secures the helix/linker contact, which acts as a latch maintaining channel closure. The modeled side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model--an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Making this residue less hydrophobic (L596A/G/W/Q/K) reduces open probability [Po; loss-offunction (LOF)], likely due to altered interactions at the polar-nonpolar interface. L596I raises Po [gain-of-function (GOF)], apparently by placing its methyl group further inward and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the levels of α-carbons of H-bonded residues L596 and W733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP helix latch, respectively. These results highlight that L596 lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4. [ABSTRACT FROM AUTHOR]

Published
2016
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37. Mechanosensitive Channels and Sensing Osmotic Stimuli in Bacteria.

Author

Martinac, Boris, Blount, Paul, Iscla, Irene, and Yuezhou Li

Abstract

Microbes are directly exposed to the elements, and one of the most acute insults they can experience is a rapid change in osmotic environment. The forces that are generated by even small changes in the osmolarity are massive. In response to increases in the osmolarity of the medium, also called osmotic upshock, the cell transports and synthesizes cytoplasmic osmoprotectants, which thus help to maintain its turgor. A subsequent severe osmotic downshock is more than an inconvenience, it is life threatening. The cell swells, its cell-wall becomes compromised, and without immediate action the organism would lyse. Largeconductance mechanosensitive channels within the cytoplasmic membrane prevent this needless death by serving as biological emergency release valves. Two such bacterial mechanosensitive channel families have been extensively studied: MscL and MscS. For each, a crystal structure of a family member has been obtained, and detailed models for structural changes that occur upon gating have been postulated. As we learn more of the molecular mechanisms by which these channels sense and respond to membrane tension, we discover similarities not only between these two relatively distant families, but also potentially with the more complex mechanosensory systems of eukaryotic organisms. [ABSTRACT FROM AUTHOR]

Published
2008
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38. Mechanosensitive Channels Gated by Membrane Tension.

Author

Kamkin, A., Kiseleva, I., Kamkin, Andre, Kiseleva, Irina, Blount, Paul, Yuezhou, Li, Moe, Paul C., and Iscla, Irene

Abstract

What are the stimuli that are sensed by mechanoreceptors? Researchers have now begun to address this question and determine the molecular mechanisms underlying channels that are gated by mechanical forces. Two quite different models now exist. The first is that the channels are ‘tethered' to cytoskeleton and or extracellular components, which thus exert forces on the channel that lead to gating. The second model predicts that the channel protein directly senses biophysical changes that occur within the membrane when it is under tension. Several lines of evidence indicate that many putative mechanosensitive channels are indeed tethered by other proteins, however in many instances the exact role this tethering plays in mechanosensing has yet to be fully clarified. On the other hand, the cloning and study of bacterial mechanosensitive channels demonstrated that channels can directly sense tension within the membrane. Evidence obtained from several of the more complex eukaryotic mechanosensory systems suggests that a number of eukaryotic channels from divergent families similarly sense tension within the membrane. [ABSTRACT FROM AUTHOR]

Published
2008
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40. An in vivo screen reveals protein-lipid interactions crucial for gating a mechanosensitive channel.

Author

Iscla, Irene, Wray, Robin, and Blount, Paul

Subjects
*PROTEINS, *HIGH-tryptophan diet, *ESCHERICHIA coli, *OSMOREGULATION, *AMINO acids, *LIPIDS
Abstract

The bacterial mechanosensitive channel MscL is the best-studied mechanosensor, thus serving as a paradigm of how a protein senses and responds to mechanical force. Models for the transition of Escherichia coli MscL from closed to open states propose a tilting of the transmembrane domains in the plane of the membrane, suggesting dynamic protein-lipid interactions. Here, we used a rapid in vivo assay to assess the function of channels that were post-translationally modified at several different sites in a region just distal to the cytoplasmic end of the second transmembrane helix. We utilized multiple probes with various affinities for the membrane environment. The in vivo functional data, combined with site-directed mutagenesis, single-channel analyses, and tryptophan fluorescence measurements, confirmed that lipid interactions within this region are critical for MscL gating. The data suggest a model in which this region acts as an anchor for the transmembrane domain tilting during gating. Furthermore, the conservation of analogous motifs among many other channels suggests a conserved protein-lipid dynamic mechanism. [ABSTRACT FROM AUTHOR]

Published
2011
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