Papers by Delphine Bichet
Frontiers in Pharmacology, 2021
Two-pore domain (K2P) potassium channels are active as dimers. They produce inhibitory currents r... more Two-pore domain (K2P) potassium channels are active as dimers. They produce inhibitory currents regulated by a variety of stimuli. Among them, TALK1, TALK2 and TASK2 form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding them are clustered on chromosomal region 6p21. They are expressed in different tissues including the pancreas. By analyzing single cell transcriptomic data, we show that these channels are co-expressed in insulin-secreting pancreatic β cells. By different approaches we show that they form functional heterodimers. Heteromerization of TALK2 with TALK1 or with TASK2 endorses TALK2 with sensitivity to extracellular alkalosis in the physiological range. The association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of their potential physiological and pathophysiological roles.
Frontiers in Pharmacology, 2021
Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, incl... more Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the “up” and “down” conformations identified by crystallography do not correspond to open and closed states of these channels.
The Journal of Physiology, 2021
Potassium channels form the largest family of ion channels with more than 80 members involved in ... more Potassium channels form the largest family of ion channels with more than 80 members involved in cell excitability and signalling. Most of them exist as homomeric channels, whereas specific conditions are required to obtain heteromeric channels. It is well established that Lamyaa Khoubza is a PhD student in biological science from University of Nice Côte-d' Azur. She is studying heteromerization of K 2P channels under the supervision of Dr Bichet. Franck Chatelain is a CNRS researcher specialized in ion channel structure and function. Sylvain Feliciangeli is an INSERM researcher specialized in trafficking of ion channels.
Biophysical Journal, 2020
Increase in relative HCO 3 À permeability (P HCO3 /P Cl) of cystic fibrosis transmembrane conduct... more Increase in relative HCO 3 À permeability (P HCO3 /P Cl) of cystic fibrosis transmembrane conductance regulator (CFTR) channel by[Cl À ] i-sensitive kinases is essential for pancreatic HCO 3 À secretion. Defects in pancreatic HCO 3 À
Frontiers in Molecular Neuroscience, 2018
TREK/TRAAK channels are polymodal K + channels that convert very diverse stimuli, including bioac... more TREK/TRAAK channels are polymodal K + channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP 2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP 2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP 2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions.
Proceedings of the National Academy of Sciences of the United States of America, Jan 28, 2016
The tandem of pore domain in a weak inwardly rectifying K(+)channel (Twik)-related acid-arachidon... more The tandem of pore domain in a weak inwardly rectifying K(+)channel (Twik)-related acid-arachidonic activated K(+)channel (TRAAK) and Twik-related K(+)channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channel...
The Journal of biological chemistry, Jan 10, 2014
Despite a high level of sequence homology, tandem pore domain halothane-inhibited K(+) channel 1 ... more Despite a high level of sequence homology, tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents, whereas THIK2 is silent. This lack of activity is due to a unique combination of intracellular retention and weak basal activity in the plasma membrane. Here, we designed THIK subunits containing dominant negative mutations (THIK1(DN) and THIK2(DN)). THIK2(DN) mutant inhibits THIK1 currents, whereas THIK1(DN) inhibits an activated form of THIK2 (THIK2-A155P-I158D). In situ proximity ligation assays and Förster/fluorescence resonance energy transfer (FRET) experiments support a physical association between THIK1 and THIK2. Next, we expressed covalent tandems of THIK proteins to obtain expression of pure heterodimers. Td-THIK1-THIK2 (where Td indicates tandem) produces K(+) currents of amplitude similar to Td-THIK1-THIK1 but with a noticeable difference in the current kinetics. Unlike Td-THIK2-THIK2 that is mainly detected in the endoplasmic retic...
Pflügers Archiv - European Journal of Physiology, 2014
Among K 2P channels, a few of them turned out to be difficult to express in heterologous systems ... more Among K 2P channels, a few of them turned out to be difficult to express in heterologous systems and were coined "silent subunits". Recent studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For TWIK1 and THIK2 channels, silence is related to a combination of intracellular retention and low intrinsic activity. TWIK1 is constitutively endocytosed from the plasma membrane before being transported to recycling endosomes, whereas THIK2 is restricted to endoplasmic reticulum. These intracellular localizations are related to trafficking signals located in the cytoplasmic parts of the channels. When these motifs are mutated or masked, channels are redistributed at the plasma membrane and produce measurable currents. However, these currents are of modest amplitude. This weak basal activity is due to a hydrophobic barrier in the deep pore that limits water and ions in the conduction pathway. Other silent channels KCNK7, TWIK2, and TASK5 are still under study. Expression and characterization of these K 2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.
Trends in Cardiovascular Medicine, 2006
European Journal of Biochemistry, 2000
Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the ... more Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4±Cys25, Cys10±Cys30, Cys14±Cys32 and Cys20±Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD 50 of 0.2 mg per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC 50 of 23 nm) and rat Kv1.2 channels (IC 50 of 0.44 nm) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125 I-labeled apamin for binding onto rat brain synaptosomes with an IC 50 of 55 pm. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK-and Kv1-types of K 1 channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.
Proceedings of the National Academy of Sciences, 2006
Potassium channels are necessary for a number of essential biological tasks such as the generatio... more Potassium channels are necessary for a number of essential biological tasks such as the generation of action potentials and setting the resting membrane potential in cells, both of which require that these channels selectively permit the passage of potassium ions while suppressing the flow of other ions. Generally, this selectivity is attributed to a narrow stretch of the channel known as the selectivity filter. Over this stretch ions are dehydrated, and the backbone oxygen atoms of the protein mimic the ion's loss of coordination by water. However, channels are long pores with spatially distinct ion-binding sites that all must be traversed during ion permeation. We have shown that selectivity of mutant Kir3.2 (GIRK2) channels can be substantially amplified by introducing acidic residues into the cavity, a binding site below the selectivity filter. Here, we carry out electrostatic calculations on homology models to quantify the degree of stabilization that these mutations have o...
Proceedings of the National Academy of Sciences, 2004
P/Q Ca 2+ channel activity is inhibited by G protein-coupled receptor activation. Channel inhibit... more P/Q Ca 2+ channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gβγ binding onto the pore-forming subunit, Ca v 2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary β 3 -subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of β 3 . Along with the observation that Gβγ has no effect on the I-V curve of Ca v 2.1 alone, we propose that the reluctant mode promoted by Gβγ corresponds to a state in which the β 3 -subunit has been displaced from its channel-binding site. We validate this hypothesis with a β 3 -I-II 2.1 loop chimera construct. Gβγ binding onto the I-II 2.1 loop portion of the chimera releases the β 3 -binding domain and makes it available for binding onto the I-II loop o...
Proceedings of the National Academy of Sciences, 2012
TWIK1 belongs to the family of background K + channels with two pore domains. In native and trans... more TWIK1 belongs to the family of background K + channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K + selective at neutral pH, it becomes permeable to Na + at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1 −/− pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.
Pflügers Archiv - European Journal of Physiology, 2010
Mechano-gated ion channels play a key physiological role in cardiac, arterial, and skeletal myocy... more Mechano-gated ion channels play a key physiological role in cardiac, arterial, and skeletal myocytes. For instance, opening of the non-selective stretch-activated cation channels in smooth muscle cells is involved in the pressure-dependent myogenic constriction of resistance arteries. These channels are also implicated in major pathologies, including cardiac hypertrophy or Duchenne muscular dystrophy. Seminal work in prokaryotes and invertebrates highlighted the role of transient receptor potential (TRP) channels in mechanosensory transduction. In mammals, recent findings have shown that the canonical TRPC1 and TRPC6 channels are key players in muscle mechanotransduction. In the present review, we will focus on the functional properties of TRPC1 and TRPC6 channels, on their mechano-gating, regulation by interacting cytoskeletal and scaffolding proteins, physiological role and implication in associated diseases.
Pflügers Archiv - European Journal of Physiology, 2007
This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-ac... more This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca 2+ permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic.
Neuron, 2000
role of the various subunits in channel functioning and assembly appears to be an absolute prereq... more role of the various subunits in channel functioning and assembly appears to be an absolute prerequisite to the understanding of VDCC physiology. The function of the auxiliary subunits seems particularly interesting, as they greatly affect channel properties. These modifica
Nature Reviews Neuroscience, 2003
Technique in which an alteration is made either at a specific site or randomly in a DNA molecule.... more Technique in which an alteration is made either at a specific site or randomly in a DNA molecule. Mutated DNA is then reintroduced into a cell and analysed with various techniques to determine which parts of a protein or nucleotide sequence are crucial for its function.
Journal of Molecular and Cellular Cardiology, 2010
Despite the central physiological importance of cardiovascular mechanotransduction, the molecular... more Despite the central physiological importance of cardiovascular mechanotransduction, the molecular identities of the sensors and the signaling pathways have long remained elusive. Indeed, how pressure is transduced into cellular excitation has only recently started to emerge. In both arterial and cardiac myocytes, the diacylglycerol-sensitive canonical transient receptor potential (TRPC) subunits are proposed to underlie the stretch-activated depolarizing cation channels. An indirect mechanism of activation through a ligandindependent conformational switch of Gq-coupled receptors by mechanical stress is invoked. Such a mechanism involving the angiotensin type 1 receptor and TRPC6 is proposed to trigger the arterial myogenic response to intraluminal pressure. TRPC6 is also involved in load-induced cardiac hypertrophy. In this review, we will focus on the molecular basis of pressure sensing in the cardiovascular system and associated disease states.
Journal of Biological Chemistry, 1999
The abbreviations used are: AID, ␣ 1 interaction domain; BID,  interaction domain; GST, glutathi... more The abbreviations used are: AID, ␣ 1 interaction domain; BID,  interaction domain; GST, glutathione S-transferase; PCR, polymerase chain reaction; MBP, maltose-binding protein; 35 S- 4 , [ 35 S]methioninelabeled  4 subunit; PAGE, polyacrylamide gel electrophoresis. Fusion proteins are referred to as, for example, GST-NT A for that containing the entire amino-terminal region of ␣ 1A , and GST-NT A,2-52 for the truncated form of this which contains only residues 2-52.
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Papers by Delphine Bichet