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Affective touch is necessary for proper neurodevelopment and sociability. However, it remains unclear how the neurons innervating the skin detect affective and social behaviors. The C low-threshold mechanoreceptors (C-LTMRs), a specific population of somatosensory neurons in mice, appear particularly well suited, physiologically and anatomically, to perceive affective and social touch. However, their contribution to sociability has not been resolved yet. Our observations revealed that C-LTMR functional deficiency induced social isolation and reduced tactile interactions in adulthood. Conversely, transient increase in C-LTMR excitability in adults, using chemogenetics, was rewarding, promoted touch-seeking behaviors, and had prosocial influences on group dynamics. This work provides the first empirical evidence that specific peripheral inputs alone can drive complex social behaviors. It demonstrates the existence of a specialized neuronal circuit, originating in the skin, wired to promote interactions with other individuals.

IF 10.848; International audience; Cerebellar atrophy is a key neuroradiological finding usually associated with cerebellar ataxia and cognitive development defect in children. Unlike the adult forms, early onset cerebellar atrophies are classically described as mostly autosomal recessive conditions and the exact contribution of de novo mutations to this phenotype has not been assessed. In contrast, recent studies pinpoint the high prevalence of pathogenic de novo mutations in other developmental disorders such as intellectual disability, autism spectrum disorders and epilepsy. Here, we investigated a cohort of 47 patients with early onset cerebellar atrophy and/or hypoplasia using a custom gene panel as well as whole exome sequencing. De novo mutations were identified in 35% of patients while 27% had mutations inherited in an autosomal recessive manner. Understanding if these de novo events act through a loss or a gain of function effect is critical for treatment considerations. To gain a better insight into the disease mechanisms causing these cerebellar defects, we focused on CACNA1G, a gene not yet associated with the early-onset form. This gene encodes the Cav3.1 subunit of T-type calcium channels highly expressed in Purkinje neurons and deep cerebellar nuclei. We identified four patients with de novo CACNA1G mutations. They all display severe motor and cognitive impairment, cerebellar atrophy as well as variable features such as facial dysmorphisms, digital anomalies, microcephaly and epilepsy. Three subjects share a recurrent c.2881G>A/p.Ala961Thr variant while the fourth patient has the c.4591A>G/p.Met1531Val variant. Both mutations drastically impaired channel inactivation properties with significantly slower kinetics (∼5 times) and negatively shifted potential for half-inactivation (>10 mV). In addition, these two mutations increase neuronal firing in a cerebellar nuclear neuron model and promote a larger window current fully inhibited by TTA-P2, a selective T-type channel blocker. This study highlights the prevalence of de novo mutations in early-onset cerebellar atrophy and demonstrates that A961T and M1531V are gain of function mutations. Moreover, it reveals that aberrant activity of Cav3.1 channels can markedly alter brain development and suggests that this condition could be amenable to treatment.

International audience; Medicinal plants represent a significant reservoir of unexplored substances for early-stage drug discovery. Of interest, two flowering Mediterranean plants have been used for thousands of years for their beneficial effects on nervous disorders, including anxiety and mood. However, the therapeutic potential of these plants regarding their ability to target ion channels and neuronal excitability remains largely unknown. Towards this goal, we have investigated the ability of Lavender and Rosemary to modulate T-type calcium channels (TTCCs). TTCCs play important roles in neuronal excitability, neuroprotection, sensory processes and sleep. These channels are also involved in epilepsy and pain. Using the whole-cell patch-clamp technique, we have characterized how Lavender and Rosemary extracts, as well as their major active compounds Linalool and Rosmarinic acid, modulate the electrophysiological properties of recombinant TTCCs (CaV3.2) expressed in HEK-293T cells. Both the methanolic and essential oil extracts as well as the active compounds of these plants inhibit Cav3.2 current in a concentration-dependent manner. In addition, these products also induce a negative shift of the steady-state inactivation of CaV3.2 current with no change in the activation properties. Taken together, our findings reveal that TTCCs are a molecular target of the Lavender and Rosemary compounds, suggesting that inhibition of TTCCs could contribute to the anxiolytic and the neuroprotective effects of these plants.

Voltage-gated Ca2+ channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca2+ ion itself. This is well exemplified by the Ca2+-dependent inactivation of L-type Ca2+ channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca2+ channels, a long-held view is that they are not regulated by intracellular Ca2+. Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca2+ channels. We demonstrate that a rise in submembrane Ca2+ induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca2+-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca2+ entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca2+ channels to their physiological roles. DOI: http://dx.doi.org/10.7554/eLife.22331.001, eLife digest Neurons, muscle cells and many other types of cells use electrical signals to exchange information and coordinate their behavior. Proteins known as calcium channels sit in the membrane that surrounds the cell and can generate electrical signals by allowing calcium ions to cross the membrane and enter the cell during electrical activities. Although calcium ions are needed to generate these electrical signals, and for many other processes in cells, if the levels of calcium ions inside cells become too high they can be harmful and cause disease. Cells have a “feedback” mechanism that prevents calcium ion levels from becoming too high. This mechanism relies on the calcium ions that are already in the cell being able to close the calcium channels. This feedback mechanism has been extensively studied in two types of calcium channel, but it is not known whether a third group of channels – known as Cav3 channels – are also regulated in this way. Cav3 channels are important in electrical signaling in neurons and have been linked with epilepsy, chronic pain and various other conditions in humans. Cazade et al. investigated whether calcium ions can regulate the activity of human Cav3 channels. The experiments show that these channels are indeed regulated by calcium ions, but using a distinct mechanism to other types of calcium channels. For the Cav3 channels, calcium ions alter the gating properties of the channels so that they are less easily activated . As a result, fewer Cav3 channels are “available” to provide calcium ions with a route into the cell. The next steps following on from this work will be to identify the molecular mechanisms underlying this new feedback mechanism. Another challenge will be to find out what role this calcium ion-driven feedback plays in neurological disorders that are linked with altered Cav3 channel activity. DOI: http://dx.doi.org/10.7554/eLife.22331.002

Primary aldosteronism (PA) is the most common form of secondary hypertension. Mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D are found in aldosterone producing adenoma (APA) and familial hyperaldosteronism (FH). A recurrent mutation in CACNA1H (coding for Cav3.2) was identified in a familial form of early onset PA. Here we performed whole exome sequencing (WES) in patients with different types of PA to identify new susceptibility genes. Four different heterozygous germline CACNA1H variants were identified. A de novo Cav3.2 p.Met1549Ile variant was found in early onset PA and multiplex developmental disorder. Cav3.2 p.Ser196Leu and p.Pro2083Leu were found in two patients with FH, and p.Val1951Glu was identified in one patient with APA. Electrophysiological analysis of mutant Cav3.2 channels revealed significant changes in the Ca2 + current properties for all mutants, suggesting a gain of function phenotype. Transfections of mutant Cav3.2 in H295R-S2 cells led to increased aldosterone production and/or expression of genes coding for steroidogenic enzymes after K+ stimulation. Identification of CACNA1H mutations associated with early onset PA, FH, and APA suggests that CACNA1H might be a susceptibility gene predisposing to PA with different phenotypic presentations, opening new perspectives for genetic diagnosis and management of patients with PA., Graphical abstract Image 8, Highlights • Germline CACNA1H mutations were identified in patients with different forms of primary aldosteronism (PA) • CACNA1H mutations modify electrophysiological channel properties • They induce increased aldosterone production and/or expression of genes coding for steroidogenic enzymes • Functional modifications are in favor of a gain of function phenotype • PA associated to CACNA1H mutations may include early onset PA with multiplex developmental disorder, familial and sporadic forms. Despite recent advances, the pathogenesis of a large proportion of sporadic and familial cases of primary aldosteronism (PA) has not been elucidated. Our work suggests that genetic variants in CACNA1H, coding for the voltage-gated T-type calcium channel Cav3.2, may be responsible for PA with different phenotypic presentations. These mutations affect the electrophysiological channel properties and increase aldosterone production and/or expression of genes coding for steroidogenic enzymes. These findings bring substantial new insight into the genetic and phenotypic spectrum of PA opening new perspectives for genetic screening for CACNA1H mutations in PA, aiming to early diagnosis and better medical care.

T-type (Cav3) calcium channels play important roles in neuronal excitability, both in normal and pathological activities of the brain. In particular, they contribute to hyper-excitability disorders such as epilepsy. Here we have characterized the anticonvulsant properties of TTA-A2, a selective T-type channel blocker, in mouse. Using the maximal electroshock seizure (MES) as a model of tonic-clonic generalized seizures, we report that mice treated with TTA-A2 (0.3 mg/kg and higher doses) were significantly protected against tonic seizures. Although no major change in Local Field Potential (LFP) pattern was observed during the MES seizure, analysis of the late post-ictal period revealed a significant increase in the delta frequency power in animals treated with TTA-A2. Similar results were obtained for Cav3.1-/- mice, which were less prone to develop tonic seizures in the MES test, but not for Cav3.2-/- mice. Analysis of extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and c-Fos expression revealed a rapid and elevated neuronal activation in the hippocampus following MES clonic seizures, which was unchanged in TTA-A2 treated animals. Overall, our data indicate that TTA-A2 is a potent anticonvulsant and that the Cav3.1 isoform plays a prominent role in mediating TTA-A2 tonic seizure protection.

Background: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. Methods: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Ca v β2 chaperone regulates channel density at the plasma membrane. Results: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Ca v α1.2 and the Akt-dependent phosphorylation status of Ca v β2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Ca v β2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Ca v α1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Ca v α1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Ca v β2, thus facilitating the chaperoning of Ca v α1.2; and promotion of Ca v α1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Ca v β2 to the nucleus, where it limits the transcription of Ca v α1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Ca v β2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. Conclusions: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Ca v β2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

International audience; Creep forming is a process where plastic deformation is applied at the material's aging temperature. It enables to obtain parts of complex shape with reduced internal stresses and finds applications, for instance, in the aerospace industry. In this article, we report in-situ small-angle X-ray scattering measurements during creep experiments carried out on an AA7449 Al-Zn-Mg-Cu alloy in the T7651 temper. In the range of temperatures of 413 K to 453 K (140 A degrees C to 180 A degrees C), we show that the initial microstructure is not stable with respect to the applied stress/strain. Accelerated precipitation coarsening is shown to occur, clearly related to the plastic deformation. This strain-induced microstructure evolution is shown to happen even at temperatures well below the aging temperature that has led to the initial temper.

International audience; A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic β-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I–II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels.

T-type calcium channels (T-channels) contribute to a wide variety of physiological functions, especially in the cardiovascular and nervous systems. Recent studies using knock-out mouse models have been instrumental in documenting further the role of T-channels in sleep, heartbeat, pain and epilepsy. Importantly, several novel aspects of the regulation of these channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Here, we review recent evidence supporting that the Cav3 subunits of T-channels are modulated by endogenous ligands such as anandamide, zinc, redox and oxidizing agents, as well as G-protein and protein kinases pathways. The study of T-channel mutations associated with childhood absence epilepsy has also revealed new aspects of Cav3 subunit trafficking. Collectively, these findings identify novel regulatory mechanisms involved in the fine tuning of T-channel expression and activity, and offer new directions for the design of novel therapeutic strategies targeting these channels.

Modulation of low voltage-activated Ca(V)3 T-type calcium channels remains poorly characterized compared with high voltage-activated Ca(V)1 and Ca(V)2 calcium channels. Notably, it is yet unresolved whether Ca(V)3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature ( approximately 30-37 degrees C) but not at room temperature ( approximately 22-27 degrees C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated Ca(V)2.3 currents. We also report that neither Ca(V)3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of Ca(V)3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of Ca(V)3 T-channels and further highlight the key role of the physiological temperature in the effects described.

International audience; Despite its expression in different cell types, transient receptor potential V2 (TRPV2) is still the most cryptic members of the TRPV channel family. 2-Aminoethoxydiphenyl borate (2APB) has been shown to be a common activator of TRPV1, TRPV2, and TRPV3, but 2APB-triggered TRPV2 activation remains to be thoroughly characterized. In this study, we have developed an assay based on cell lines stably expressing mouse TRPV2 channels and intracellular calcium measurements to perform a pharmacological profiling of the channel. Phenyl borate derivatives were found to activate mouse TRPV2 with similar potencies and thus were used to screen a panel of channel blockers. Besides the classic TRP inhibitors ruthenium red (RR) and 1-(beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF96365), two potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine, and an inhibitor of capacitative calcium entry, 1-(2-(trifluoromethyl) phenyl) imidazole (TRIM), were found to inhibit TRPV2 activation by 100 microM 2APB. Activation by 300 microM 2APB, however, could only be inhibited by RR and TRIM. Electrophysiological recordings demonstrated that TEA inhibition was use-dependent, suggesting that high concentrations of 2APB might induce a progressive conformational change of the channel. Comparison of TRPV2 orthologs revealed that the human channel was insensitive to 2APB. Analysis of chimeric constructs of mouse and human TRPV2 channels showed that the molecular determinants of 2APB sensitivity could be localized to the intracellular amino and carboxyl domains. Finally, using lentiviral-driven expression, we demonstrate that hTRPV2 exerts a dominant-negative effect on 2APB activation of native rodent TRPV2 channels and thus may provide an interesting tool to investigate cellular functions of TRPV2 channels.

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.

Zinc (Zn2+) functions as a signalling molecule in the nervous system and modulates many ionic channels. In this study, we have explored the effects of Zn2+ on recombinant T-type calcium channels (CaV3.1, CaV3.2 and CaV3.3). Using tsA-201 cells, we demonstrate that CaV3.2 current (IC50, 0.8 μm) is significantly more sensitive to Zn2+ than are CaV3.1 and CaV3.3 currents (IC50, 80 μm and ∼160 μm, respectively). This inhibition of CaV3 currents is associated with a shift to more negative membrane potentials of both steady-state inactivation for CaV3.1, CaV3.2 and CaV3.3 and steady-state activation for CaV3.1 and CaV3.3 currents. We also document changes in kinetics, especially a significant slowing of the inactivation kinetics for CaV3.1 and CaV3.3, but not for CaV3.2 currents. Notably, deactivation kinetics are significantly slowed for CaV3.3 current (∼100-fold), but not for CaV3.1 and CaV3.2 currents. Consequently, application of Zn2+ results in a significant increase in CaV3.3 current in action potential clamp experiments, while CaV3.1 and CaV3.2 currents are significantly reduced. In neuroblastoma NG 108-15 cells, the duration of CaV3.3-mediated action potentials is increased upon Zn2+ application, indicating further that Zn2+ behaves as a CaV3.3 channel opener. These results demonstrate that Zn2+ exhibits differential modulatory effects on T-type calcium channels, which may partly explain the complex features of Zn2+ modulation of the neuronal excitability in normal and disease states.

Low-voltage-activated T-type calcium channels are expressed in various tissues, especially in the brain, where they promote neuronal firing and are involved in slow wave sleep and absence epilepsy. While the transduction pathways by which hormones and neurotransmitters modulate high-voltage-activated calcium channels are beginning to be unraveled, those implicated in T-type calcium channel regulation remain obscure. Several neurotransmitters and hormones regulate native T-type calcium channels, although some contradictory data have been reported depending on the cell type studied. This review focuses on the short-term (minutes range) modulation of T-type calcium channels by neurotransmitters and hormones and on the roles of G proteins and protein kinases in these modulatory effects. Results obtained in different native tissues are discussed and compared with the more recent studies of the three cloned T-type calcium channels CaV3.1, CaV3.2 and CaV3.3 in expression systems.

TREK-1 (KCNK2) is a K(2P) channel that is highly expressed in fetal neurons. This K(+) channel is opened by a variety of stimuli, including membrane stretch and cellular lipids. Here, we show that the expression of TREK-1 markedly alters the cytoskeletal network and induces the formation of actin- and ezrin-rich membrane protrusions. The genetic inactivation of TREK-1 significantly alters the growth cone morphology of cultured embryonic striatal neurons. Cytoskeleton remodelling is crucially dependent on the protein kinase A phosphorylation site S333 and the interactive proton sensor E306, but is independent of channel permeation. Conversely, the actin cytoskeleton tonically represses TREK-1 mechano-sensitivity. Thus, the dialogue between TREK-1 and the actin cytoskeleton might influence both synaptogenesis and neuronal electrogenesis.

Lysophosphatidic acid (LPA) is an abundant cellular lipid with a myriad of biological effects. It plays an important role in both inter- and intracellular signaling. Activation of the LPA1-3 G-protein-coupled receptors explains many of the extracellular effects of LPA, including cell growth, differentiation, survival, and motility. However, LPA also acts intracellularly, activating the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma that regulates gene transcription. This study shows that the novel subfamily of mechano-gated K2P channels comprising TREK-1, TREK-2, and TRAAK is strongly activated by intracellular LPA. The LPA-activated 2P domain K+ channels are intracellular ligand-gated K+ channels such as the Ca2+- or the ATP-sensitive K+ channels. LPA reversibly converts these mechano-gated, pH- and voltage-sensitive channels into leak conductances. Gating conversion of the 2P domain K+ channels by intracellular LPA represents a novel form of ion channel regulation. Thus, the TREK and TRAAK channels should be included in the LPA-associated physiological and disease states.

Calcium influx via low-voltage activated alpha(1H) (Ca(v)3.2) T-currents participates in the morphological and electrical differentiation of neuroblastoma NG108-15 cells. We investigated whether an autocrine mechanism could contribute to this differentiation process. The presence of factors secreted by NG108-15 cells was identified through the use of conditioned media (CM) obtained from differentiated cells. These CM significantly increased neuritogenesis with no change in the HVA calcium channel expression. CM-induced neuritogenesis persists during alpha(1H) current block, whereas CM obtained from cells transfected with an alpha(1H) antisense did not induce neuritogenesis. These data indicate that morphological differentiation of NG108-15 cells depends on an autocrine mechanism, which is controlled by alpha(1H) currents. Such a mechanism is likely to play a role in the various differentiation processes that imply alpha(1H) T-type Ca(2+) channels.

Le role de l’ion Ca2+ dans la signalisation cellulaire est largement reconnu. Les canaux calciques de type T (canaux T) constituent une voie d’entree de calcium tres particuliere dans la cellule car ils presentent des caracteristiques specifiques sur le plan fonctionnel. Ces canaux sont actives pour de tres faibles depolarisations membranaires. Ils peuvent egalement induire une entree importante de calcium en generant un courant de fenetre durant des periodes tres precises du developpement, ainsi que dans certaines situations physiopathologiques. Les hypotheses concernant le role physiologique et physiopathologique des canaux de type T dans l’evolution du phenotype cellulaire sont nombreuses: proliferation cellulaire, differenciation cellulaire, activation de genes, secretion d’hormones,... mais restent encore mal definies dans le contexte des etudes actuelles. Recemment, la generation d’outils moleculaires specifiques suite au clonage des ADN complementaires de ces canaux a permis de demontrer leur role dans la differenciation neuronale. Les donnees recentes obtenues par notre equipe qui indiquent une implication des canaux T generes par la sous-unite α1H dans la differenciation neuronale sont presentees et discutees dans cet article.

Neuronal differentiation involves both morphological and electrophysiological changes, which depend on calcium influx. Voltage-gated calcium channels (VGCCs) represent a major route for calcium entry into neurons. The recently cloned low-voltage-activated T-type calcium channels (T-channels) are the first class of VGCCs functionally expressed in most developing neurons, as well as in neuroblastoma cell lines, but their roles in neuronal development are yet unknown. Here, we document the part played by T-channels in neuronal differentiation. Using NG108-15, a cell line that recapitulates early steps of neuronal differentiation, we demonstrate that blocking T-currents by nickel, mibefradil, or the endogenous cannabinoid anandamide prevents neuritogenesis without affecting neurite outgrowth. Similar results were obtained using antisense oligodeoxynucleotides directed against the alpha1H T-channel subunit. Furthermore, we describe that inhibition of alpha1H T-channel activity impairs concomitantly, but independently, both high-voltage-activated calcium channel expression and neuritogenesis, providing strong evidence for a dual role of T-channels in both morphological and electrical changes at early stages of neuronal differentiation.

In several types of neurons, firing is an intrinsic property produced by specific classes of ion channels. Low-voltage-activated T-type calcium channels (T-channels), which activate with small membrane depolarizations, can generate burst firing and pacemaker activity. Here we have investigated the specific contribution to neuronal excitability of cloned human T-channel subunits. Using HEK-293 cells transiently transfected with the human alpha(1G) (Ca(V)3.1), alpha(1H) (Ca(V)3.2) and alpha(1I) (Ca(V)3.3) subunits, we describe significant differences among these isotypes in their biophysical properties, which are highlighted in action potential clamp studies. Firing activities occurring in cerebellar Purkinje neurons and in thalamocortical relay neurons used as voltage clamp waveforms revealed that alpha(1G) channels and, to a lesser extent, alpha(1H) channels produced large and transient currents, while currents related to alpha(1I) channels exhibited facilitation and produced a sustained calcium entry associated with the depolarizing after-potential interval. Using simulations of reticular and relay thalamic neuron activities, we show that alpha(1I) currents contributed to sustained electrical activities, while alpha(1G) and alpha(1H) currents generated short burst firing. Modelling experiments with the NEURON model further revealed that the alpha(1G) channel and alpha(1I) channel parameters best accounted for T-channel activities described in thalamocortical relay neurons and in reticular neurons, respectively. Altogether, the data provide evidence for a role of alpha(1I) channel in pacemaker activity and further demonstrate that each T-channel pore-forming subunit displays specific gating properties that account for its unique contribution to neuronal firing.

T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation via its metabolism leading to prostanoids, leukotrienes, and epoxyeicosatrienoic acids (EETs). However, the effects of these latter molecules on T-currents have not been investigated. Here, we describe the effects of the major cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products on the three human recombinant T-channels (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3), as compared to those of AA. We identified the P450 epoxygenase product, 5,6-EET, as a potent physiological inhibitor of Ca(v)3 currents. The effects of 5,6-EET were observed at sub-micromolar concentrations (IC50 = 0.54 mu M), occurred in the minute range, and were reversible. The 5,6-EET inhibited the Ca(v)3 currents at physiological resting membrane potentials mostly by inducing a large negative shift in their steady-state inactivation properties. Using knockout mice for Ca(v)3.1 and Ca(v)3.2, we demonstrated that the vasodilation of preconstricted mesenteric arteries induced by 5,6-EET was specifically impaired in Ca(v)3.2 knockout mice. Overall, our results indicate that inhibition of Ca(v)3 currents by 5,6-EET is an important mechanism controlling the vascular tone.

This chapter discusses the regulation of the mechano-gated K(2P) channel, TREK-1 by membrane phospholipids. TREK-1 (KCNK2 or K2P2.1) is a polymodal K(+) channel that is activated by membrane stretch, intracellular acidosis, heat, and cellular lipids, such as arachidonic acid (AA). Phospholipids, including PIP2, exert a dual dose-dependent effect on TREK-1. Low concentrations transform the mechanogated K(+) channel TREK-1 into a leak K(+) channel. The phospholipid-sensing domain is a positively charged cluster in the proximal C-terminal domain. This region also encompasses the proton sensor E306 that is required for the activation of TREK-1 by cytosolic acidosis. Protonation of E306 increases channel-phospholipid interaction leading to TREK-1 opening without direct-mechanical stimulation. At higher concentrations, intracellular phospholipids inhibit channel activation by stretch, intracellular acidosis, and AA. Binding endogenous negative inner leaflet phospholipids with polylysine reduces the inhibition and reveals channel stimulation by exogenous intracellular phospholipids. Both stimulatory and inhibitory effects are observed with phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA), but not diacylglycerol (DG), suggesting that the phosphate at position 3 is required, although the net charge is not critical. Membrane phospholipids, including PIP2, are major regulators of TREK-1 channel activity.

Voltage dependent L-Type calcium-channels (LTCCs) are located on the cardiomyocyte membrane and regulate cardiac contraction and rhythmicity. In human pathologies, such as heart failure (HF), decreased inward calcium current (I Ca ) is frequently observed. Here, we generated a mimetic peptide (MP) that targets LTCCs and restores impaired intracellular calcium homeostasis through a novel mechanism. Effective delivery of MP, fused with a cell penetrating peptide, was found to correct Ca2+ alterations in a mouse model of HF, in human cardiomyocytes derived from induced pluripotent stem-cells. These data provide a proof-of-concept supporting a therapeutic role for MP to treat human diseases related to LTCC abnormalities. Category: heart failure biology

International audience; To exert its analgesic action, paracetamol requires complex metabolism to produce a brain-specific lipoamino acid compound, AM404, which targets central transient receptor potential vanilloid receptors (TRPV1). Lipoamino acids are also known to induce analgesia through T-type calcium-channel inhibition (Cav3.2). In this study we show that the antinociceptive effect of paracetamol in mice is lost when supraspinal Cav3.2 channels are inhibited. Therefore, we hypothesized a relationship between supraspinal Cav3.2 and TRPV1, via AM404, which mediates the analgesic effect of paracetamol. AM404 is able to activate TRPV1 and weakly inhibits Cav3.2. Interestingly, activation of TRPV1 induces a strong inhibition of Cav3.2 current. Supporting this, intracerebroventricular administration of AM404 or capsaicin produces antinociception that is lost in Cav3.2−/− mice. Our study, for the first time, 1) provides a molecular mechanism for the supraspinal antinociceptive effect of paracetamol; 2) identifies the relationship between TRPV1 and the v3.2 channel">Cav3.2 channel; and 3) suggests supraspinal Cav3.2 inhibition as a potential pharmacological strategy to alleviate pain.

International audience; The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE) and calcium (EEEE) selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM) was lowered to 0.1 mM and were inhibited (\textgreater40% to \textgreater90%) with 10 microM Gd3+, 100 microM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (\textless30%) to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 microM mibefradil or 100 microM nifedipine. We hypothesize that the rapid changes in leak current size in response to changing external cations or drugs relates to their influences on the membrane seal adherence and the electro-osmotic flow of mobile cations channeling in crevices of a particular pore size in the interface between the negatively charged patch electrode and the lipid membrane. Observed sodium leak conductance currents in weak patch seals are reproducible between the electrode glass interface with cell membranes, artificial lipid or Sylgard rubber.

Low-voltage-activated or T-type Ca(2+) channels (T-channels) are widely expressed, especially in the central nervous system where they contribute to pacemaker activities and are involved in the pathogenesis of epilepsy. Proper elucidation of their cellular functions has been hampered by the lack of selective pharmacology as well as the absence of generic endogenous regulations. We report here that both cloned (alpha(1G), alpha(1H) and alpha(1I) subunits) and native T-channels are blocked by the endogenous cannabinoid, anandamide. Anandamide, known to exert its physiological effects through cannabinoid receptors, inhibits T-currents independently from the activation of CB1/CB2 receptors, G-proteins, phospholipases and protein kinase pathways. Anandamide appears to be the first endogenous ligand acting directly on T-channels at submicromolar concentrations. Block of anandamide membrane transport by AM404 prevents T-current inhibition, suggesting that anandamide acts intracellularly. Anandamide preferentially binds and stabilizes T-channels in the inactivated state and is responsible for a significant decrease of T-currents associated with neuronal firing activities. Our data demonstrate that anandamide inhibition of T-channels can regulate neuronal excitability and account for CB receptor-independent effects of this signaling molecule.

The recently cloned T-type calcium channel alpha1I (Cav3.3) displays atypically slow kinetics when compared to native T-channels. Possible explanations might involve alternative splicing of the alpha1I subunit, or the use of expression systems that do not provide a suitable environment (auxiliary subunit, phosphorylation, glycosylation...). In this study, two human alpha1I splice variants, the alpha1I-a and alpha1I-b isoforms that harbour distinct carboxy-terminal regions were studied using various expression systems. As the localization of the alpha1I subunit is primarily restricted to neuronal tissues, its functional expression was conducted in the neuroblastoma/glioma cell line NG 108-15, and the results compared to those obtained in HEK-293 cells and Xenopus oocytes. In Xenopus oocytes, both isoforms exhibited very slow current kinetics compared to those obtained in HEK-293 cells, but the alpha1I-b isoform generated faster currents than the alpha1I-a isoform. Both activation and inactivation kinetics of alpha1I currents were significantly faster in NG 108-15 cells, while deactivating tail currents were two times slower, compared to those obtained in HEK-293 cells. Moreover, the alpha1-b isoform showed significantly slower deactivation kinetics both in NG 1080-15 and in HEK-293 cells. Altogether, these data emphasize the advantage of combining several expression systems to reveal subtle differences in channel properties and further indicate that the major functional differences between both human alpha1I isoforms are related to current kinetics. More importantly, these data suggest that the expression of the alpha1I subunit in neuronal cells contributes to the "normalization" of current kinetics to the more classical, fast-gated T-type Ca2+ current.

At least three genes encode T-type calcium channel alpha(1) subunits, and identification of cDNA transcripts provided evidence that molecular diversity of these channels can be further enhanced by alternative splicing mechanisms, especially for the alpha(1G) subunit (Ca(V)3.1). Using whole-cell patch-clamp procedures, we have investigated the electrophysiological properties of five isoforms of the human alpha(1G) subunit that display a distinct III-IV linker, namely, alpha(1G-a), alpha(1G-b), and alpha(1G-bc), as well as a distinct II-III linker, namely, alpha(1G-ae), alpha(1G-be), as expressed in HEK-293 cells. We report that insertion e within the II-III linker specifically modulates inactivation, steady-state kinetics, and modestly recovery from inactivation, whereas alternative splicing within the III-IV linker affects preferentially kinetics and voltage dependence of activation, as well as deactivation and inactivation. By using voltage-clamp protocols mimicking neuronal activities, such as cerebellar train of action potentials and thalamic low-threshold spike, we describe that inactivation properties of alpha(1G-a) and alpha(1G-ae) isoforms can support channel behaviors reminiscent to those described in native neurons. Altogether, these data demonstrate that expression of distinct variants for the T-type alpha(1G) subunit can account for specific low-voltage-activated currents observed in neuronal tissues.

We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.

T-type calcium channels (T-channels/CaV3) have unique biophysical properties allowing a calcium influx at resting membrane potential of most cells. T-channels are ubiquitously expressed in many tissues and contribute to low-threshold spikes and burst firing in central neurons as well as to pacemaker activities in cardiac cells. They also emerged as potential targets to treat cancer and hypertension. Regulation of these channels appears complex, and several studies have indicated that CaV3.1, CaV3.2, and CaV3.3 currents are directly inhibited by multiple endogenous lipids independently of membrane receptors or intracellular pathways. These bioactive lipids include arachidonic acid and ω3 poly-unsaturated fatty acids; the endocannabinoid anandamide and other N-acylethanolamides; the lipoamino-acids and lipo-neurotransmitters; the P450 epoxygenase metabolite 5,6-epoxyeicosatrienoic acid; as well as similar molecules with 18–22 carbons in the alkyl chain. In this review, we summarize evidence for direct effects of these signaling molecules, the molecular mechanisms underlying the current inhibition, and the involved chemical features. The impact of this modulation in physiology and pathophysiology is discussed with a special emphasis on pain aspects and vasodilation. Overall, these data clearly indicate that T-current inhibition is an important mechanism by which bioactive lipids mediate their physiological functions.

T-type calcium channels (T/Ca(v)3-channels) are implicated in various physiologic and pathophysiologic processes such as epilepsy, sleep disorders, hypertension, and cancer. T-channels are the target of endogenous signaling lipids including the endocannabinoid anandamide, the ω3-fatty acids, and the lipoamino-acids. However, the precise molecular mechanism by which these molecules inhibit T-current is unknown. In this study, we provided a detailed electrophysiologic and pharmacologic analysis indicating that the effects of the major N-acyl derivatives on the Ca(v)3.3 current share many similarities with those of TTA-A2 [(R)-2-(4-cyclopropylphenyl)-N-(1-(5-(2,2,2-trifluoroethoxy)pyridin-2-yl)ethyl)acetamide], a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [(3)H]TTA-A1 [(R)-2-(4-(tert-butyl)phenyl)-N-(1-(5-methoxypyridin-2-yl)ethyl)acetamide], we demonstrated that polyunsaturated lipids, which inhibit the Ca(v)3.3 current, as NAGly (N-arachidonoyl glycine), NASer (N-arachidonoyl-l-serine), anandamide, NADA (N-arachidonoyl dopamine), NATau (N-arachidonoyl taurine), and NA-5HT (N-arachidonoyl serotonin), all displaced [(3)H]TTA-A1 binding to membranes prepared from cells expressing Ca(v)3.3, with Ki in a micromolar or submicromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca(v)3.3 current, had no effect on [(3)H]TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca(v)3.3 current. In addition, TTA-Q4 [(S)-4-(6-chloro-4-cyclopropyl-3-(2,2-difluoroethyl)-2-oxo-1,2,3,4-tetrahydroquinazolin-4-yl)benzonitrile], a positive allosteric modulator of [(3)H]TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Ca(v)3.3 current. Overall, our results demonstrate a common molecular mechanism for the synthetic T-channel inhibitors and the endogenous lipids, and indicate that TTA-A2 and TTA-Q4 could be important pharmacologic tools to dissect the involvement of T-current in the physiologic effects of endogenous lipids.

International audience; Ligand-gated ion channels are prototypic oligomeric membrane proteins whose stoichiometry determines their functional properties and subcellular localization. Deciphering the quaternary structure of such protein complexes is an arduous task and usually requires the combination of multiple approaches. ATP-gated P2X receptors are formed by the association of three subunits, but the quaternary arrangement of the seven P2X subunits at the plasma membrane remains poorly characterized. By combining bioluminescence resonance energy transfer, bifunctional fluorescence complementation and protein biochemistry, we developed an experimental approach that allows precise determination of rat P2X receptor quaternary assembly. We found that P2X5 subunits associate with P2X1, P2X2, and P2X4 subunits. We demonstrate that P2X5 and P2X2 subunits interact to form as yet uncharacterized heteromeric receptors with alternate stoichiometries, both present at the plasma membrane. P2X2/5 receptors display functional properties such as pore dilatation, membrane blebbing, and phosphatidylserine exposure that were previously thought to be characteristic hallmarks of the P2X7 receptor. In mouse, P2X2 and P2X5 subunits colocalize and physically interact in specific neuronal populations suggesting that other P2X receptors might contribute to cellular responses typically attributed to P2X7 receptor.

International audience; The coupling between precipitation and plasticity has been systematically investigated in an Al-Zn-Mg-Cu alloy using in situ small-angle X-ray scattering measurements during thermomechanical tests. Material pre-aged to two different initial precipitate conditions has been examined. Each pre-aged condition has been strained at 160 degrees C and we show that the plasticity induces an accelerated coarsening kinetics, which we characterize in terms of the evolution of the precipitate size. This acceleration is correlated with the degree of plastic strain, but does not depend markedly on strain rate. The experimental data strongly suggests that the accelerated kinetics is mainly linked with the accumulation of a supersaturation of vacancies during plastic flow that increases the effective diffusion constant. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Alteration in the density or function of L-Type Calcium Channels (LTCCs) has been related to cardiovascular diseases such as heart failure and diabetic cardiomyopathy. It could therefore be envisioned that increasing LTCC density may improve cardiac function in heart failure. Recently, we determined that the Ser-Thr kinase Akt plays a key role in regulating cardiac inotropism through the modulation of LTCC density and function. Specifically, we found that the LTCC pore-forming channel subunit Cava1.2 contains highly evolutionary conserved PEST sequences (signals for rapid proteolytic degradation) that are responsible for direct Cava1.2 protein degradation. Phosphorylation of the C-terminal coiled coil of the Cavb2 chaperone subunit enhances LTCC protein stability by preventing PEST-mediated Cava1.2 degradation. The aim of this study was to further dissect this Akt-dependent fine-tuning mechanism regulating LTCC density, searching for potential Akt-phosphomimetic (APM) molecules that could enhance LTCC density. Using yeast two-hybrid screening, we found that APM Cavb2 sequences interact with the globular domain of Cavb2 itself in a solvent-exposed region that we named Tail Interacting Domain (TID). Biochemical and functional assays as well as site-specific mutagenesis in TID identified the minimal aminoacid sequence responsible for the TID-tail interaction. In addition, through an approach comprising western blot analyses, fluorescent-based calcium assays, calcium current (ICaL) measurements, molecular modeling and peptide arrays, we identified the minimal APMs that efficiently protects Cava1.2 from protein degradation. Based on our in vitro results, we suggest that the identified APM sequence/peptide could be used as a “therapeutic approach”; for increasing or reestablishing impaired cardiac contractility in mouse models of cardiomyopathy in which the LTCC density is altered, thus enhancing inotropism.

T-type calcium channels, i.e. Cav3.1, Cav3.2 and Cav3.3 channels, have important roles in cell excitability and calcium signalling and contribute to a wide variety of physiological functions especially in nervous system. Over the past few years, several endogenous ligands regulating Cav3 activity were identified, including bioactive lipids such as the endocannabinoid anandamide (N-arachidonoyl ethanolamine). We now provide evidence that the T-type / Cav3 calcium channels are potently and reversibly inhibited by various lipoamino acids, including N-arachidonoyl glycine (NAGly, IC50 ∼ 600 nM for Cav3.2) and N-arachidonoyl 3-OH-gamma-aminobutyric acid (NAGABA-OH, IC50 ∼200 nM for Cav3.2). This inhibition involves a large shift in the Cav3.2 steady-state inactivation and persists during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. It appears that lipoamino acids are the most active endogenous ligand family acting on T-channels. Importantly, lipoamino acids have weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents as well as on TRPV1 and TASK1 currents. These data indicate that lipoamino acid effects may be selective of T-type channels over HVA calcium channels, sodium channels as well as the anandamide-sensitive TRPV1 and TASK1 channels. It also suggests that these ligands can modulate multiple cell functions via T-type calcium channel regulation. In line with this, we found that lipoamino acids evoke a thermal analgesia in wild-type but not in Cav3.2 KO mice. Collectively, our data identify lipoamino acids as a new potent and selective family of endogenous T-type channel inhibitors.

Lipoamino acids are anandamide-related endogenous molecules that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacological targets underlying their physiological effects. Various lipoamino acids, includingN-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC50∼200 nmforN-arachidonoyl 3-OH-γ-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addition, we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.

Genomic mining, the process of sifting through billions of genomic and EST sequences of several different species has led to the molecular identification of a family of low voltage activating channels, more commonly referred to as T-type channels. Historically, these channels were initially identified through the use of the patch-damp technique on various neuronal preparations. They were characterized by their small conductance, rapid voltage-dependent inactivation, a small window current and slow deactivation kinetics and their remarkable property of being able to open at membrane potentials just above the resting membrane potential of neurons. This property would allow for the entry of Ca2+ without the initiation of an action potential triggered by the opening of sodium channels. Thus the activity of these channels would contribute to modifying membrane excitability, allowing Ca2+ signaling events to occur at subthreshold potentials, and potentially modulate waveform patterns.

This chapter discusses the regulation of the mechano-gated K(2P) channel, TREK-1 by membrane phospholipids. TREK-1 (KCNK2 or K2P2.1) is a polymodal K(+) channel that is activated by membrane stretch, intracellular acidosis, heat, and cellular lipids, such as arachidonic acid (AA). Phospholipids, including PIP2, exert a dual dose-dependent effect on TREK-1. Low concentrations transform the mechanogated K(+) channel TREK-1 into a leak K(+) channel. The phospholipid-sensing domain is a positively charged cluster in the proximal C-terminal domain. This region also encompasses the proton sensor E306 that is required for the activation of TREK-1 by cytosolic acidosis. Protonation of E306 increases channel-phospholipid interaction leading to TREK-1 opening without direct-mechanical stimulation. At higher concentrations, intracellular phospholipids inhibit channel activation by stretch, intracellular acidosis, and AA. Binding endogenous negative inner leaflet phospholipids with polylysine reduces the inhibition and reveals channel stimulation by exogenous intracellular phospholipids. Both stimulatory and inhibitory effects are observed with phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA), but not diacylglycerol (DG), suggesting that the phosphate at position 3 is required, although the net charge is not critical. Membrane phospholipids, including PIP2, are major regulators of TREK-1 channel activity.

Anandamide, originally described as an endocannabinoid, is the main representative molecule of a new class of signaling lipids including endocannabinoids and N-acyl-related molecules, eicosanoids, and fatty acids. Bioactive lipids regulate neuronal excitability by acting on G-protein-coupled receptors (such as CB1) but also directly modulate various ionic conductances including voltage-activated T-type calcium channels (T-channels). However, little is known about the properties and the specificity of this new class of molecules on their various targets. In this study, we have investigated the chemical determinants involved in anandamide-induced inhibition of the three cloned T-channels: Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3. We show that both the hydroxyl group and the alkyl chain of anandamide are key determinants of its effects on T-currents. As follows, T-currents are also inhibited by fatty acids. Inhibition of the three Ca(V)3 currents by anandamide and arachidonic acid does not involve enzymatic metabolism and occurs in cell-free inside-out patches. Inhibition of T-currents by fatty acids and N-acyl ethanolamides depends on the degree of unsaturation but not on the alkyl chain length and consequently is not restricted to eicosanoids. Inhibition increases for polyunsaturated fatty acids comprising 18-22 carbons when cis-double bonds are close to the carboxyl group. Therefore the major natural (food-supplied) and mammalian endogenous fatty acids including gamma-linolenic acid, mead acid, and arachidonic acid as well as the fully polyunsaturated omega3-fatty acids that are enriched in fish oil eicosapentaenoic and docosahexaenoic acids are potent inhibitors of T-currents, which possibly contribute to their physiological functions.