Lavender oil-potent anxiolytic properties via modulating voltage dependent calcium channels

Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety

The role of subunit composition on prepulse facilitation of the cardiac L-type calcium channel

AbstractFacilitation of calcium current by depolarizing prepulses has been observed in many cells including cardiac muscle. The mechanism underlying prepulse facilitation is controversial with respect to the requirements of channel subunits and cAMP kinase. We found that coexpression of the cardiac α1C-a subunit with the cardiac β2a subunit significantly promotes the facilitation of IBa by strong depolarizing prepulses. The magnitude of IBa facilitation depended on the voltage potential of the prepulse and the interval duration between prepulse and test pulse. Prepulse facilitation was not affected by coexpression of AKAP79 and conditions favoring cAMP-dependent phosphorylation. Prepulse facilitation was also observed in cells expressing an α1C-a subunit which was truncated at residue 1733 removing the cAMP kinase site at Ser-1928. Facilitation was abolished by coexpression of the α2δ-1 or α2δ-3 subunit. We conclude that the expressed α1C-a β2a complex is sufficient to support prepulse facilitation. Facilitation is prevented by coexpression of the α2δ subunit

Extracellular Ca2+ Modulates the Effects of Protons on Gating and Conduction Properties of the T-type Ca2+ Channel α1G (CaV3.1)

Since Ca2+ is a major competitor of protons for the modulation of high voltage–activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel α1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca2+. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca2+-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca2+. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca2+ and Na+. Mutation of the EEDD pore locus altered the Ca2+-dependent proton effects on channel selectivity and permeation. We conclude that Ca2+ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel

Pore Structure Influences Gating Properties of the T-type Ca2+ Channel α1G

The selectivity filter of all known T-type Ca2+ channels is built by an arrangement of two glutamate and two aspartate residues, each one located in the P-loops of domains I–IV of the α1 subunit (EEDD locus). The mutations of the aspartate residues to glutamate induce changes in the conduction properties, enhance Cd2+ and proton affinities, and modify the activation curve of the channel. Here we further analyze the role of the selectivity filter in the gating mechanisms of T-type channels by comparing the kinetic properties of the α1G subunit (CaV3.1) to those of pore mutants containing aspartate-to-glutamate substitution in domains III (EEED) or IV (EEDE). The change of the extracellular pH induced similar effects on the activation properties of α1G and both pore mutants, indicating that the larger affinity of the mutant channels for protons is not the cause of the gating modifications. Both mutants showed alterations in several gating properties with respect to α1G, i.e., faster macroscopic inactivation in the voltage range from −10 to 50 mV, positive voltage shift and decrease in the voltage sensitivity of the time constants of activation and deactivation, decrease of the voltage sensitivity of the steady-state inactivation, and faster recovery from inactivation for long repolarization periods. Kinetic modeling suggests that aspartate-to-glutamate mutations in the EEDD locus of α1G modify the movement of the gating charges and alter the rate of several gating transitions. These changes are independent of the alterations of the selectivity properties and channel protonation

The two-pore channel TPC1 is required for efficient protein processing through early and recycling endosomes

Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and pathogenicity of Ebola viruses. Here, we investigate the cellular function of TPC1 using protein toxins as model substrates for distinct endosomal processing routes. Toxin uptake and activation through early endosomes but not processing through other compartments were reduced in TPC1 knockout cells. Detailed co-localization studies with subcellular markers confirmed predominant localization of TPC1 to early and recycling endosomes. Proteomic analysis of native TPC1 channels finally identified direct interaction with a distinct set of syntaxins involved in fusion of intracellular vesicles. Together, our results demonstrate a general role of TPC1 for uptake and processing of proteins in early and recycling endosomes, likely by providing high local Ca2+ concentrations required for SNARE-mediated vesicle fusion

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites

Two-Pore Channels Regulate Expression of Various Receptors and Their Pathway-Related Proteins in Multiple Ways

Two-pore channels (TPCs) constitute a small family of ion channels within membranes of intracellular acidic compartments, such as endosomes and lysosomes. They were shown to provide transient and locally restricted Ca2+-currents, likely responsible for fusion and/or fission events of endolysosomal membranes and thereby for intracellular vesicle trafficking. Genetic deletion of TPCs not only affects endocytosis, recycling, and degradation of various surface receptors but also uptake and impact of bacterial protein toxins and entry and intracellular processing of some types of viruses. This review points to important examples of these trafficking defects on one part but mainly focuses on the resulting impact of the TPC inactivation on receptor expression and receptor signaling. Thus, a detailed RNA sequencing analysis using TPC1-deficient fibroblasts uncovered a multitude of changes in the expression levels of surface receptors and their pathway-related signaling proteins. We refer to several classes of receptors such as EGF, TGF, and insulin as well as proteins involved in endocytosis

Control of intestinal motility by the Ca 1.2 L‐type calcium channel in mice

Wegener J, Schulla V, Koller A, et al. Control of intestinal motility by the Ca 1.2 L‐type calcium channel in mice. The FASEB Journal. 2006;20(8):1260-1262