Multiple modalities converge on a common gate to control K2P channel function

K2P potassium channels play important roles in the regulation of neuronal excitability. K2P channels are gated chemical, thermal, and mechanical stimuli, and the present study identifies and characterizes a common molecular gate that responds to all different stimuli, both activating and inhibitory ones

Two MscS Homologs Provide Mechanosensitive Channel Activities in the Arabidopsis Root

In bacterial and animal systems, mechanosensitive (MS) ion channels are thought to mediate the perception of pressure, touch, and sound 1, 2 and 3. Although plants respond to a wide variety of mechanical stimuli, and although many mechanosensitive channel activities have been characterized in plant membranes by the patch-clamp method, the molecular nature of mechanoperception in plant systems has remained elusive [4]. Likely candidates are relatives of MscS (Mechanosensitive channel of small conductance), a well-characterized MS channel that serves to protect E. coli from osmotic shock [5]. Ten MscS-Like (MSL) proteins are found in the genome of the model flowering plant Arabidopsis thaliana 4, 6 and 7. MSL2 and MSL3, along with MSC1, a MscS family member from green algae, are implicated in the control of organelle morphology 8 and 9. Here, we characterize MSL9 and MSL10, two MSL proteins found in the plasma membrane of root cells. We use a combined genetic and electrophysiological approach to show that MSL9 and MSL10, along with three other members of the MSL family, are required for MS channel activities detected in protoplasts derived from root cells. This is the first molecular identification and characterization of MS channels in plant membranes

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account

A Human TREK-1/HEK Cell Line: A Highly Efficient Screening Tool for Drug Development in Neurological Diseases

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model

Electrical Remodelling in Cardiac Disease

The human heart responds to various diseases with structural, mechanical, and electrical remodelling processes [...

Du canal dépendant du voltage AtVDAC-1 à l'identification de deux canaux mécano-sensibles MSL9 et MSL10 sur la membrane plasmique d'arabidopsis thaliana

Les travaux présentés dans ce documents caractérisent l activité de trois canaux ioniques. La protéine AtVDAC-1, une VDAC (Voltage Dependent Anion Channel) atypique de par sa double localisation mitochondrie/membrane plasmique est étudiée en système artificiel. Nous montrons qu elle forme un canal possédant les grandes caractéristiques biophysiques des VDAC avec cependant quelques singularités comme par exemple une sensibilité au voltage asymétrique et décalée. Les recherches de cette activité en système homologue, sur la membrane de protoplastes racinaires d Arabidopsis thaliana, nous ont conduit à la mise en évidence d une activité mécano-sensible (MS). Sa caractérisation fait l objet de la seconde partie de ce document. Suite à l analyse réalisée en patch-clamp de différents mutants et de l expression transitoire de gènes candidats en système homologue nous identifions les protéines MSL9 et MSL10 ( Mechanosensitive channel of Small conductance Like), comme étant deux canaux MS. Ces deux protéines, homologues du canal bactérien MscS (Mechanosensitive channel of Small conductance), s activent en réponse à une augmentation de la tension de membrane et présentent des conductances distinctes. Nos travaux montrent que ces canaux sont perméants aux ions chlorure et nitrate et non aux ions calcium. Nous suggérons que l activité MS dominante sur les protoplastes racinaires de plantules de génotype sauvage résulte du fonctionnement en complexe de ces deux protéines . MSL9 et MSL10 sont les premiers canaux MS identifiés génétiquement et fonctionnellement sur une plante supérieure.The activities of three ion channels were characterized in this thesis. AtVDAC-1, a voltage Dependent Anion Channel an unusual localization, both in mitochondria and in the plasma membrane, was studied in an artificial system. It forms a channel with biophysical features of VDAC with some peculiarities like a nonsymetrical voltage dependency. Searching for the AtVDAC-1 activity on the membrane of root protoplasts from Arabidopsis thaliana led to the identification of a mechanosensitive activity. Its characterization is the subject of the second part of this document. Different mutants and candidate genes transiently expressed in homologous system were analysed using the patch-clamp technique. From the experiments, the proteins named MSL9 and MSL10 (Mechanosensitive channel of Small conductance Like) are identified as two mechanosensitive channels. These two proteins, homologs of the bacterial MscS (Mechano Sensitive channel of Small conductance), are activated in reponse to an increase in membrane tension and have distinct conductances. Our experiments showed that these channels are permeant for chloride and nitrate ions and not for calcium. We suggest that the main mechanosensitive activity in root protoplasts of wild-type mechanosensitive channels functionally and genetically identified in higher plants.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

AtMSL9 and AtMSL10: Sensors of plasma membrane tension in Arabidopsis roots

Plant cells, like those of animals and bacteria, are able to sense physical deformation of the plasma membrane. Mechanosensitive (MS) channels are proteins that transduce mechanical force into ion flux, providing a mechanism for the perception of mechanical stimuli such as sound, touch and osmotic pressure. We recently identified AtMSL9 and AtMSL10, two mechanosensitive channels in Arabidopsis thaliana, as molecular candidates for mechanosensing in higher plants.1 AtMSL9 and AtMSL10 are members of a family of proteins in Arabidopsis that are related to the bacterial MS channel MscS, termed MscS-Like (or MSL).2 MscS (Mechanosensitive channel of Small conductance) is one of the best-characterized MS channels, first identified as an electrophysiological activity in the plasma membrane (PM) of giant E. coli spheroplasts.3,4 Activation of MscS is voltage-independent, but responds directly to tension applied to the membrane and does not require other cellular proteins for this regulation.5,6 MscS family members are widely distributed throughout bacterial and archaeal genomes, are present in all plant genomes yet examined, and are found in selected fungal genomes.2,7,8 MscS homolgues have not yet been identified in animals

R type anion channel: a multifunctional channel seeking its molecular identity.

International audiencePlant genomes code for channels involved in the transport of cations, anions and uncharged molecules through membranes. Although the molecular identity of channels for cations and uncharged molecules has progressed rapidly in the recent years, the molecular identity of anion channels has lagged behind. Electrophysiological studies have identified S-type (slow) and R-type (rapid) anion channels. In this brief review, we summarize the proposed functions of the R-type anion channels which, like the S-type, were first characterized by electrophysiology over 20 years ago, but unlike the S-type, have still yet to be cloned. We show that the R-type channel can play multiple roles