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1.
J Cannabis Res ; 2(1): 17, 2020 May 11.
Article in English | MEDLINE | ID: mdl-33526117

ABSTRACT

BACKGROUND: Cannabis contains Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) as the primary constituents responsible for pharmacological activity. However, there are numerous additional chemically-related structures to Δ9-THC and CBD that are pharmacologically active and may influence the pharmacological properties of Δ9-THC and CBD. This study chemically characterized the cannabinoid constituents in a series of cannabis chemovar extracts and investigated the potential cannabinoid entourage effect in two behavioral assays. METHODS: Six chemovar extracts were compared to pure Δ9-THC, CBD and morphine for effects on the following behavioral assays in mice: hot plate and tail suspension. The battery of behavioral tests was conducted post intravenous administration of cannabis chemovar extract. Cannabinoid profiles of extracts were analyzed using high performance liquid chromatography. Cannabis extracts were administered at equal doses of Δ9-THC to investigate the role of their cannabinoid profiles in modulating the effects of Δ9-THC. Dose response curves were fit using a log[inhibitor] vs response three parameter model and differences between group means were determined using a one-way ANOVA followed by a post hoc test. RESULTS: Cannabis chemovars tested in this study exhibited substantially different cannabinoid profiles. All chemovars produced dose-dependent immobility in the tail suspension assay and dose-dependent antinociception in the hot plate assay. The maximum antinociceptive effect and ED50 was comparable between cannabis chemovars and Δ9-THC. Two cannabis chemovars produced significantly greater immobility in the tail suspension test, with no significant differences in ED50. CONCLUSIONS: Commercially available cannabis chemovars vary widely in cannabinoid content, but when equalized for Δ9-THC content, they produce similar behavioral effects with two exceptions. These findings provide only limited support for the entourage hypothesis. Further studies are necessary to characterize the nature of these pharmacological differences between cannabis chemovars and pure Δ9-THC.

2.
Front Mol Neurosci ; 10: 41, 2017.
Article in English | MEDLINE | ID: mdl-28286469

ABSTRACT

Hyperpolarization-activated cyclic nucleotide-gated "HCN" channels, which underlie the hyperpolarization-activated current (Ih), have been proposed to play diverse roles in neurons. The presynaptic HCN channel is thought to both promote and inhibit neurotransmitter release from synapses, depending upon its interactions with other presynaptic ion channels. In larvae of Drosophila melanogaster, inhibition of the presynaptic HCN channel by the drug ZD7288 reduces the enhancement of neurotransmitter release at motor terminals by serotonin but this drug has no effect on basal neurotransmitter release, implying that the channel does not contribute to firing under basal conditions. Here, we show that genetic disruption of the sole HCN gene (Ih) reduces the amplitude of the evoked response at the neuromuscular junction (NMJ) of third instar larvae by decreasing the number of released vesicles. The anatomy of the (NMJ) is not notably affected by disruption of the Ih gene. We propose that the presynaptic HCN channel is active under basal conditions and promotes neurotransmission at larval motor terminals. Finally, we demonstrate that Ih partial loss-of-function mutant adult flies have impaired locomotion, and, thus, we hypothesize that the presynaptic HCN channel at the (NMJ) may contribute to coordinated movement.

3.
Am J Physiol Cell Physiol ; 298(5): C1066-76, 2010 May.
Article in English | MEDLINE | ID: mdl-20130205

ABSTRACT

All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary trajectories that diverged in an isoform-specific manner after gene duplication from a common HCN ancestor that lacked N-glycosylation and was able to localize efficiently to the cell surface.


Subject(s)
Cell Membrane/metabolism , Cyclic Nucleotide-Gated Cation Channels/genetics , Cyclic Nucleotide-Gated Cation Channels/metabolism , Ion Channels/metabolism , Potassium Channels/genetics , Potassium Channels/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , CHO Cells , Cricetinae , Cricetulus , Embryo, Mammalian/metabolism , Evolution, Molecular , Gene Expression Regulation , Glycosylation , Heart/embryology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Ion Channels/genetics , Mice , Molecular Sequence Data , Myocardium/metabolism , Phylogeny , Rats
4.
Proc Natl Acad Sci U S A ; 103(8): 2886-91, 2006 Feb 21.
Article in English | MEDLINE | ID: mdl-16477030

ABSTRACT

Voltage-gated channels maintain cellular resting potentials and generate neuronal action potentials by regulating ion flux. Here, we show that Ether-à-go-go (EAG) K+ channels also regulate intracellular signaling pathways by a mechanism that is independent of ion flux and depends on the position of the voltage sensor. Regulation of intracellular signaling was initially inferred from changes in proliferation. Specifically, transfection of NIH 3T3 fibroblasts or C2C12 myoblasts with either wild-type or nonconducting (F456A) eag resulted in dramatic increases in cell density and BrdUrd incorporation over vector- and Shaker-transfected controls. The effect of EAG was independent of serum and unaffected by changes in extracellular calcium. Inhibitors of p38 mitogen-activated protein (MAP) kinases, but not p44/42 MAP kinases (extracellular signal-regulated kinases), blocked the proliferation induced by nonconducting EAG in serum-free media, and EAG increased p38 MAP kinase activity. Importantly, mutations that increased the proportion of channels in the open state inhibited EAG-induced proliferation, and this effect could not be explained by changes in the surface expression of EAG. These results indicate that channel conformation is a switch for the signaling activity of EAG and suggest an alternative mechanism for linking channel activity to the activity of intracellular messengers, a role that previously has been ascribed only to channels that regulate calcium influx.


Subject(s)
Drosophila Proteins/physiology , Ether-A-Go-Go Potassium Channels/physiology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Biological Transport , Cell Proliferation/drug effects , Cells, Cultured , Drosophila/genetics , Drosophila Proteins/genetics , Ether-A-Go-Go Potassium Channels/genetics , Fibroblasts/enzymology , Fibroblasts/metabolism , Ions/metabolism , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Myoblasts/enzymology , Myoblasts/metabolism , NIH 3T3 Cells , Protein Kinase Inhibitors/pharmacology , Signal Transduction , Transfection , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
5.
J Neurosci ; 25(20): 4898-907, 2005 May 18.
Article in English | MEDLINE | ID: mdl-15901771

ABSTRACT

Signaling complexes are essential for the modulation of excitability within restricted neuronal compartments. Adaptor proteins are the scaffold around which signaling complexes are organized. Here, we demonstrate that the Camguk (CMG)/CASK adaptor protein functionally modulates Drosophila Ether-á-go-go (EAG) potassium channels. Coexpression of CMG with EAG in Xenopus oocytes results in a more than twofold average increase in EAG whole-cell conductance. This effect depends on EAG-T787, the residue phosphorylated by calcium- and calmodulin-dependent protein kinase II (Wang et al., 2002). CMG coimmunoprecipitates with wild-type and EAG-T787A channels, indicating that T787, although necessary for the effect of CMG on EAG current, is not required for the formation of the EAG-CMG complex. Both CMG and phosphorylation of T787 increase the surface expression of EAG channels, and in COS-7 cells, EAG recruits CMG to the plasma membrane. The interaction of EAG with CMG requires a noncanonical Src homology 3-binding site beginning at position R1037 of the EAG sequence. Mutation of basic residues, but not neighboring prolines, prevents binding and prevents the increase in EAG conductance. Our findings demonstrate that membrane-associated guanylate kinase adaptor proteins can modulate ion channel function; in the case of CMG, this occurs via an increase in the surface expression and phosphorylation of the EAG channel.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/physiology , Drosophila Proteins/physiology , Ether-A-Go-Go Potassium Channels/physiology , Analysis of Variance , Animals , Biotinylation/methods , Blotting, Western/methods , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Chlorocebus aethiops , Dose-Response Relationship, Radiation , Drosophila , Drosophila Proteins/genetics , Electric Stimulation/methods , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression/physiology , Immunohistochemistry/methods , Immunoprecipitation/methods , Membrane Potentials/genetics , Membrane Potentials/radiation effects , Molecular Biology/methods , Molecular Sequence Data , Mutagenesis/physiology , Mutation/physiology , Oocytes , Patch-Clamp Techniques/methods , Phosphorylation , Protein Transport/genetics , Protein Transport/physiology , RNA, Messenger/biosynthesis , Radioligand Assay/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Analysis, Protein/methods , Transfection/methods , Xenopus , src Homology Domains/physiology
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