Concept: Ion channel
The TRPM8 channel is a principal cold transducer that is expressed on some primary afferents of the somatic and cranial sensory systems. However, it is uncertain whether TRPM8-expressing afferent neurons have the ability to convey innocuous and noxious cold stimuli with sensory discrimination between the two sub-modalities. Using rat dorsal root ganglion (DRG) neurons and the patch-clamp recording technique, we characterized membrane and action potential properties of TRPM8-expressing DRG neurons at 24°C and 10°C. TRPM8-expressing neurons could be classified into TTX-sensitive (TTXs/TRPM8) and TTX-resistant (TTXr/TRPM8) subtypes based on the sensitivity to tetrodotoxin (TTX) block of their action potentials. These two subtypes of cold-sensing cells displayed different membrane and action potential properties. Voltage-activated inward Na+ currents were highly susceptible to cooling temperature and abolished by ~95% at 10°C in TTXs/TRPM8 DRG neurons, but remained substantially large at 10°C in TTXr/TRPM8 cells. In both TTXs/TRPM8 and TTXr/TRPM8 cells, voltage-activated outward K+ currents were substantially inhibited at 10°C, and the cooling-sensitive outward currents resembled A-type K+ currents. TTXs/TRPM8 neurons and TTXr/TRPM8 neurons were shown to fire action potentials at innocuous and noxious cold temperatures respectively, demonstrating sensory discrimination between innocuous and noxious cold by the two subpopulations of cold-sensing DRG neurons. The effects of cooling temperatures on voltage-gated Na+ channels and A-type K+ currents are likely to be contributing factors to sensory discrimination of cold by TTXs/TRPM8 and TTXr/TRPM8 afferent neurons.
Lubeluzole, which acts on various targets in vitro, including voltage-gated sodium channels (NaChs), was initially proposed as neuroprotectant. Lubeluzole structure contains a benzothiazole moiety (R-like) related to riluzole and a phenoxy-propranol-amine moiety (A-core) recalling propranolol. Both riluzole and propranolol are efficient NaCh blockers. We studied in detail the effects of lubeluzole (racemic mixture and single isomers), aforementioned lubeluzole moieties, and riluzole on NaChs to increase our knowledge about drug-channel molecular interactions. Compounds were tested on hNav1.4 NaChs, and F1586C or Y1593C mutants functionally expressed in HEK293 cells, using patch-clamp. Lubeluzole blocked NaChs with a remarkable effectiveness. No stereoselectivity was found. Compared to mexiletine, dissociation constant for inactivated channels was ≈600 times lower (≈11 nM), conferring to lubeluzole a huge use-dependence of great therapeutic value. The F1586C mutation impaired use-dependent block only partially, suggesting that additional amino acids are critically involved in high-affinity binding. Lubeluzole moieties were modest NaCh blockers. Riluzole blocked NaChs efficiently but lacked use-dependence, similarly to R-like. F1586C fully abolished A-core use-dependence, suggesting that A-core binds to the local anesthetic receptor. Thus lubeluzole likely binds to the local anesthetic receptor through its phenoxy-propranol-amine moiety, with consequent use-dependent behavior. Nevertheless, compared to other known NaCh blockers, lubeluzole adds a third pharmacophoric point through its benzothiazole moiety, that greatly enhances high-affinity binding and use-dependent block. If sufficient isoform specificity can be attained, the huge use-dependent block may help in the development of new NaCh inhibitors to provide pharmacotherapy for membrane excitability disorders, such as myotonia, epilepsy, or chronic pain.
- Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
- Published almost 6 years ago
Identifying the determinants of neuronal energy consumption and their relationship to information coding is critical to understanding neuronal function and evolution. Three of the main determinants are cell size, ion channel density, and stimulus statistics. Here we investigate their impact on neuronal energy consumption and information coding by comparing single-compartment spiking neuron models of different sizes with different densities of stochastic voltage-gated Na(+) and K(+) channels and different statistics of synaptic inputs. The largest compartments have the highest information rates but the lowest energy efficiency for a given voltage-gated ion channel density, and the highest signaling efficiency (bits spike(-1)) for a given firing rate. For a given cell size, our models revealed that the ion channel density that maximizes energy efficiency is lower than that maximizing information rate. Low rates of small synaptic inputs improve energy efficiency but the highest information rates occur with higher rates and larger inputs. These relationships produce a Law of Diminishing Returns that penalizes costly excess information coding capacity, promoting the reduction of cell size, channel density, and input stimuli to the minimum possible, suggesting that the trade-off between energy and information has influenced all aspects of neuronal anatomy and physiology.Journal of Cerebral Blood Flow & Metabolism advance online publication, 19 June 2013; doi:10.1038/jcbfm.2013.103.
The venom of predatory marine cone snails mainly contains a diverse array of unique bioactive peptides commonly referred to as conopeptides or conotoxins. These peptides have proven to be valuable pharmacological probes and potential drugs because of their high specificity and affinity to important ion channels, receptors and transporters of the nervous system. Most previous studies have focused specifically on the conopeptides from piscivorous and molluscivorous cone snails, but little attention has been devoted to the dominant vermivorous species.
Endophilin A2 Influences Volume-Regulated Chloride Current by Mediating ClC-3 Trafficking in Vascular Smooth Muscle Cells
- Circulation journal : official journal of the Japanese Circulation Society
- Published over 2 years ago
Previous research has demonstrated that ClC-3 is responsible for volume-regulated Cl(-)current (ICl.vol) in vascular smooth muscle cells (VSMCs). However, it is still not clear whether and how ClC-3 is transported to cell membranes, resulting in alteration ofICl.vol.Methods and Results:Volume-regulated chloride current (ICl.vol) was recorded by whole-cell patch clamp recording, and Western blotting and co-immunoprecipitation were performed to examine protein expression and protein-protein interaction. Live cell imaging was used to observe ClC-3 transporting. The results showed that an overexpression of endophilin A2 could increaseICl.vol, while endophilin A2 knockdown decreasedICl.vol. In addition, the SH3 domain of endophilin A2 mediated its interaction with ClC-3 and promotes ClC-3 transportation from the cytoplasm to cell membranes. The regulation of ClC-3 channel activity was also verified in basilar arterial smooth muscle cells (BASMCs) isolated from endophilin A2 transgenic mice. Moreover, endophilin A2 increase VSMCs proliferation induced by endothelin-1 or hypo-osmolarity.
Repeated Wi-Fi studies show that Wi-Fi causes oxidative stress, sperm/testicular damage, neuropsychiatric effects including EEG changes, apoptosis, cellular DNA damage, endocrine changes, and calcium overload. Each of these effects are also caused by exposures to other microwave frequency EMFs, with each such effect being documented in from 10 to 16 reviews. Therefore, each of these seven EMF effects are established effects of Wi-Fi and of other microwave frequency EMFs. Each of these seven is also produced by downstream effects of the main action of such EMFs, voltage-gated calcium channel (VGCC) activation. While VGCC activation via EMF interaction with the VGCC voltage sensor seems to be the predominant mechanism of action of EMFs, other mechanisms appear to have minor roles. Minor roles include activation of other voltage-gated ion channels, calcium cyclotron resonance and the geomagnetic magnetoreception mechanism. Five properties of non-thermal EMF effects are discussed. These are that pulsed EMFs are, in most cases, more active than are non-pulsed EMFs; artificial EMFs are polarized and such polarized EMFs are much more active than non-polarized EMFs; dose-response curves are non-linear and non-monotone; EMF effects are often cumulative; and EMFs may impact young people more than adults. These general findings and data presented earlier on Wi-Fi effects were used to assess the Foster and Moulder (F&M) review of Wi-Fi. The F&M study claimed that there were seven important studies of Wi-Fi that each showed no effect. However, none of these were Wi-Fi studies, with each differing from genuine Wi-Fi in three distinct ways. F&M could, at most conclude that there was no statistically significant evidence of an effect. The tiny numbers studied in each of these seven F&M-linked studies show that each of them lack power to make any substantive conclusions. In conclusion, there are seven repeatedly found Wi-Fi effects which have also been shown to be caused by other similar EMF exposures. Each of the seven should be considered, therefore, as established effects of Wi-Fi.
Targeting KCa1.1 channels with a scorpion venom peptide for the therapy of rat models of rheumatoid arthritis
- The Journal of pharmacology and experimental therapeutics
- Published about 1 year ago
Fibroblast-like synoviocytes (FLS) are a key cell-type involved in rheumatoid arthritis (RA) progression. We previously identified the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) as a regulator of FLS and that KCa1.1 inhibition reduces disease severity in RA animal models. However, systemic KCa1.1 block causes multiple side effects and in this study, we aimed to determine whether the KCa1.1 β1-3-specific venom peptide blocker iberiotoxin (IbTX) reduces disease severity in animal models of RA without inducing major side effects. We used immunohistochemistry to identify IbTX-sensitive KCa1.1 subunits in joints of rats with a model of RA. Patch clamp and functional assays were used to determine if IbTX can regulate FLS through targeting KCa1.1. We then tested the efficacy of IbTX in ameliorating disease in two rat models of RA. Finally, we determined if IbTX causes side-effects including incontinence or tremors in rats, compared to those treated with the small molecule KCa1.1 blocker paxilline. IbTX-sensitive subunits of KCa1.1 are expressed by FLS in joints of rats with experimental arthritis. IbTX inhibits KCa1.1 channels expressed by FLS from patients with RA and by FLS from rat models of RA and reduces FLS invasiveness. IbTX significantly reduces disease severity in two rat models of RA. Unlike paxilline, IbTX does not induce tremors or incontinence in rats. Overall, IbTX inhibits KCa1.1 channels on FLS and treats rat models of RA without inducing side effects associated with non-specific KCa1.1 blockade and could become the basis for the development of a new treatment for RA.
Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1)-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiology, TRPV1 knock-out (KO), and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5) carrying a Cre-dependent channelrhodopsin-2 (ChR2)-enhanced yellow fluorescent protein (eYFP) expression cassette under the control of the two neuronal POMC enhancers (nPEs). Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake.
The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+) and calcium (Ca2+) currents and potentiation of net potassium (K+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.
The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K(+)) channel to enable K(+) recycling coupled to transepithelial chloride ion (Cl(-)) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K(+) recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.”