Laboratories of Excellence

Science and, France

Laboratories of Excellence

Science and, France
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Levitz J.,University of California at Berkeley | Royal P.,University of Nice Sophia Antipolis | Royal P.,French National Center for Scientific Research | Royal P.,French Institute of Health and Medical Research | And 16 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2016

Twik-related K+ channel 1(TREK1), TREK2, and Twik-related arachidonic-acid stimulated K+ channel (TRAAK) form the TREK subfamily of two-pore-domain K+ (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.


Wang S.,University of Virginia | Wang S.,Hebei Medical University | Benamer N.,University of Nice Sophia Antipolis | Benamer N.,Laboratories of Excellence | And 13 more authors.
Journal of Neuroscience | Year: 2013

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H+ via an unidentified pH-sensitive background K+ channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K+ channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or β-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n=58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2-/- mice (n-49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2-/- mice selected based on β-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K+ currents were reduced in amplitude in RTN neurons from TASK-2-/- mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart- brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2-/- mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold. © 2013 the authors.


Bayliss D.A.,University of Virginia | Barhanin J.,University of Nice Sophia Antipolis | Barhanin J.,Laboratories of Excellence | Gestreau C.,Aix - Marseille University | Guyenet P.G.,University of Virginia
Pflugers Archiv European Journal of Physiology | Year: 2015

A number of the subunits within the family of K2P background K+ channels are sensitive to changes in extracellular pH in the physiological range, making them likely candidates to mediate various pH-dependent processes. Based on expression patterns within several brainstem neuronal cell groups that are believed to function in CO2/H+ regulation of breathing, three TASK subunits—TASK-1, TASK-2, and TASK-3—were specifically hypothesized to contribute to this central respiratory chemoreflex. For the acid-sensitive TASK-1 and TASK-3 channels, despite widespread expression at multiple levels within the brainstem respiratory control system (including presumptive chemoreceptor populations), experiments in knockout mice provided no evidence for their involvement in CO2 regulation of breathing. By contrast, the alkaline-activated TASK-2 channel has a more restricted brainstem distribution and was localized to the Phox2b-expressing chemoreceptor neurons of the retrotrapezoid nucleus (RTN). Remarkably, in a Phox2b27Ala/+ mouse genetic model of congenital central hypoventilation syndrome (CCHS) that is characterized by reduced central respiratory chemosensitivity, selective ablation of Phox2b-expressing RTN neurons was accompanied by a corresponding loss of TASK-2 expression. Furthermore, genetic deletion of TASK-2 blunted RTN neuronal pH sensitivity in vitro, reduced alkaline-induced respiratory network inhibition in situ and diminished the ventilatory response to CO2/H+ in vivo. Notably, a subpopulation of RTN neurons from TASK-2−/− mice retained their pH sensitivity, at least in part due to a residual pH-sensitive background K+ current, suggesting that other mechanisms (and perhaps other K2P channels) for RTN neuronal pH sensitivity are yet to be identified. © 2014, Springer-Verlag Berlin Heidelberg.


Tauber P.,University of Regensburg | Penton D.,University of Nice Sophia Antipolis | Penton D.,Laboratories of Excellence | Stindl J.,University of Regensburg | And 9 more authors.
Endocrinology | Year: 2014

Somatic mutations of the potassium channel KCNJ5 are found in 40% of aldosterone producing adenomas (APAs). APA-related mutations of KCNJ5 lead to a pathological Na+ permeability and a rise in cytosolic Ca 2+, the latter presumably by depolarizing the membrane and activating voltage-gated Ca2+ channels. The aim of this study was to further investigate the effects of mutated KCNJ5 channels on intracellular Na + and Ca2+ homeostasis in human adrenocortical NCI-H295R cells. Expression of mutant KCNJ5 led to a 2-fold increase in intracellular Na+ and, in parallel, to a substantial rise in intracellular Ca 2+. The increase in Ca2+ appeared to be caused by activation of voltage-gated Ca2+ channels and by an impairment of Ca2+ extrusion by Na+/Ca2+ exchangers. The mutated KCNJ5 exhibited a pharmacological profile that differed from the one of wild-type channels. Mutated KCNJ5 was less Ba2+ and tertiapin-Q sensitive but was inhibited by blockers of Na+ and Ca 2+-transporting proteins, such as verapamil and amiloride. The clinical use of these drugs might influence aldosterone levels in APA patients with KCNJ5 mutations. This might implicate diagnostic testing of APAs and could offer new therapeutic strategies. Copyright © 2014 by the Endocrine Society.


Lalli E.,French National Center for Scientific Research | Lalli E.,University of Nice Sophia Antipolis | Barhanin J.,French National Center for Scientific Research | Barhanin J.,Laboratories of Excellence | And 3 more authors.
Trends in Endocrinology and Metabolism | Year: 2016

Primary aldosteronism (PA) is caused by excessive production of aldosterone by the adrenal cortex and is determined by a benign aldosterone-producing adenoma (APA) in a significant proportion of cases. Local mechanisms, as opposed to circulatory ones, that control aldosterone production in the adrenal cortex are particularly relevant in the physiopathological setting and in the pathogenesis of PA. A breakthrough in our understanding of the pathogenetic mechanisms in APA has been the identification of somatic mutations in genes controlling membrane potential and intracellular calcium concentrations. However, recent data show that the processes of nodule formation and aldosterone hypersecretion can be dissociated in pathological adrenals and suggest a model envisaging different molecular events for the pathogenesis of APA. © 2016 Elsevier Ltd.


Comoglio Y.,University of Nice Sophia Antipolis | Comoglio Y.,French National Center for Scientific Research | Comoglio Y.,French Institute of Health and Medical Research | Comoglio Y.,Laboratories of Excellence | And 11 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K+channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phos-pholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.


Bandulik S.,University of Regensburg | Tauber P.,University of Regensburg | Lalli E.,University of Nice Sophia Antipolis | Barhanin J.,University of Nice Sophia Antipolis | And 2 more authors.
Pflugers Archiv European Journal of Physiology | Year: 2015

The physiological control of steroid hormone secretion from the adrenal cortex depends on the function of potassium channels. The “two-pore domain K+ channels” (K2P) TWIK-related acid sensitive K+ channel 1 (TASK1), TASK3, and TWIK-related K+ channel 1 (TREK1) are strongly expressed in adrenocortical cells. They confer a background K+ conductance to these cells which is important for the K+ sensitivity as well as for angiotensin II and adrenocorticotropic hormone-dependent stimulation of aldosterone and cortisol synthesis. Mice with single deletions of the Task1 or Task3 gene as well as Task1/Task3 double knockout mice display partially autonomous aldosterone synthesis. It appears that TASK1 and TASK3 serve different functions: TASK1 affects cell differentiation and prevents expression of aldosterone synthase in the zona fasciculata, while TASK3 controls aldosterone secretion in glomerulosa cells. TREK1 is involved in the regulation of cortisol secretion in fasciculata cells. These data suggest that a disturbed function of K2P channels could contribute to adrenocortical pathologies in humans. © 2014, The Author(s).


PubMed | University of Zürich, University of Mannheim, University of Ulm, University of Regensburg and Laboratories of Excellence
Type: | Journal: Respiratory physiology & neurobiology | Year: 2016

TASK-1 potassium channels have been implicated in central and peripheral chemoreception; however, the precise contribution of TASK-1 for the control of respiration is still under debate. Here, we investigated the respiration of unrestrained adult and neonatal TASK-1 knockout mice (TASK-1


Sandoz G.,University of Nice Sophia Antipolis | Sandoz G.,Laboratories of Excellence | Levitz J.,University of California at Berkeley
Frontiers in Molecular Neuroscience | Year: 2013

Optogenetic tools were originally designed to target specific neurons for remote control of their activity by light and have largely been built around opsin-based channels and pumps. These naturally photosensitive opsins are microbial in origin and are unable to mimic the properties of native neuronal receptors and channels. Over the last 8 years, photoswitchabletethered ligands (PTLs) have enabled fast and reversible control of mammalian ion channels, allowing optical control of neuronal activity. One such PTL, MAQ, contains a maleimide (M) to tether the molecule to a genetically engineered cysteine, a photoisomerizable azobenzene (A) linker and a pore-blocking quaternary ammonium group (Q). MAQ was originally used to photo-control SPARK, an engineered light-gated potassium channel derived from Shaker. Potassium channel photo-block by MAQ has recently been extended to a diverse set of mammalian potassium channels including channels in the voltage-gated and K2P families. Photoswitchable potassium channels, which maintain native properties, pave the way for the optical control of specific aspects of neuronal function and for high precision probing of a specific channel's physiological functions. To extend optical control to natively expressed channels, without overexpression, one possibility is to develop a knock-in mouse in which the wild type channel gene is replaced by its light-gated version. Alternatively, the recently developed photoswitchable-conditional-subunit technique (PCS) provides photocontrol of the channel of interest by molecular replacement of wild type complexes. Finally, photochromic ligands (PCLs) also allow photocontrol of potassium channels without genetic manipulation using soluble compounds. In this review we discuss different techniques for optical control of native potassium channels and their associated advantages and disadvantages. © 2013 Sandoz and Levitz.


Jaafari N.,French Institute of Health and Medical Research | Jaafari N.,Joseph Fourier University | Jaafari N.,Laboratories of Excellence | De Waard M.,French Institute of Health and Medical Research | And 4 more authors.
Biophysical Journal | Year: 2014

The current understanding of Ca2+ channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca2+ currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca2+ channels during physiological changes of membrane potential in complex cells, since the voltage-clamp configuration constrains the membrane potential to a given value. Here, we report for the first time to our knowledge that Ca2+ currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca2+ imaging with low-affinity indicators. The optical measurement of the Ca2+ current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca2+ channels along the apical dendrite of the CA1 hippocampal pyramidal neuron during the back-propagation of an action potential. To validate the method, we analyzed the voltage dependence of high- and low-voltage-gated Ca2+ channels. In particular, we measured the Ca2+ current component mediated by T-type channels, and we investigated the mechanisms of recovery from inactivation of these channels. This method is expected to become a reference approach to investigate Ca2+ channels in their native physiological environment. © 2014 by the Biophysical Society.

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