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At least 505 records · Page 28Linked to original sources
PubMed2000

Characteristics of P2X7 receptors from human B lymphocytes expressed in Xenopus oocytes.

Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.

Adenosine Triphosphate↗
PubMed1999

Practical limits on the maximal speed of solution exchange for patch clamp experiments.

Studying ligand-gated ion channels often requires the ability to change solutions quickly. Using finite element models, I have examined the practical limitations of how fast solutions can be exchanged on an outside-out patch using a dual stream switcher. The primary factors controlling the speed of response are the flow velocity, proximity of the patch to the exit ports, the width of the partition between the two streams, the velocity with which the streams can be moved across the patch, and the viscosity of the solutions. The practical limit seems to be a rise time of approximately 20 microseconds. The rate-limiting step is the velocity of the (usually piezo) motor that translates the streams across the patch. Increasing the perfusate viscosity improves speed by slowing dissipation of the concentration gradients. A flow switcher can also be used for bipolar temperature jumps with a rise time of approximately 100 microseconds.

Biophysical Phenomena↗
PubMed1998

Molecular cloning of cDNA encoding the alpha unit of CNGC gene from human fetal heart.

Cyclic nucleotide-gated ion channels (CNGCs) play crucial roles in visual and olfactory signal transduction. As a first step to explore the presence of a CNGC gene in human heart, we cloned a human heart CNGC gene. The sequence consists of 111 bp 5' non-coding region and a 2064 bp open reading frame which is followed by a 459 bp 3' non-coding region. The predicted protein consists of 688 amino acids with a short highly charged segment rich in lysine and glutamate. Sequence comparison indicates that the human heart cDNA is almost identical to the retinal rod photo receptor CNGC cDNA. However, the human cardiac cDNA is lacking a 205 bp Alu fragment in the 5'-uncoding region, has a glutamic acid residue at amino acid position 129, and has a replacement of glutamic acid with a lysine residue at amino acid position 99. Data obtained with northern blot analysis confirm the presence of RNA for the CNGC alpha chain. This channel might play a role in cyclic nucleotide-mediated cellular processes, such as the inotropic effect in the heart.

Amino Acid Sequence↗
PubMed1999

Sense and specificity: a molecular identity for nociceptors.

Recent cloning efforts have identified families of ligand- or voltage-gated ion channels that are expressed by pain-sensing primary afferent neurons. Pharmacological, electrophysiological and genetic studies are beginning to reveal how these signaling molecules specify roles for subsets of sensory neurons in the pain pathway.

Acids↗
PubMed1997

Patch clamp detection in capillary electrophoresis.

We describe a capillary electrophoresis-patch clamp (CE-PC) analysis of biomolecules that activate ligand-gated ion channels. CE-PC offers a powerful means for identifying receptor ligands based on the combination of the characteristic receptor responses they evoke and their differential electrophoretic migration rates. Corner frequencies, membrane reversal potentials, and mean and unitary single-channel receptor responses were calculated from currents recorded with patch clamp detection. This information was then combined with the electrophoretic mobility of the receptor ligand, which is proportional to the charge-to-frictional-drag ratio of that species. We applied CE-PC to separate and detect the endogenous receptor agonists gamma-aminobutyrate and L-glutamate and the synthetic glutamate receptor agonists N-methyl-D-aspartate and kainic acid. We present dose-response data for electrophoretically separated kainic acid and discuss its implications for making the CE-PC detection system quantitative.

Animals↗
PubMed2006

Kinetics of anesthetic-induced conformational transitions in a four-alpha-helix bundle protein.

Inhaled anesthetics are thought to alter the conformational states of Cys-loop ligand-gated ion channels (LGICs) by binding within discrete cavities that are lined by portions of four alpha-helical transmembrane domains. Because Cys-loop LGICs are complex molecules that are notoriously difficult to express and purify, scaled-down models have been used to better understand the basic molecular mechanisms of anesthetic action. In this study, stopped-flow fluorescence spectroscopy was used to define the kinetics with which inhaled anesthetics interact with (Aalpha(2)-L1M/L38M)(2), a four-alpha-helix bundle protein that was designed to model anesthetic binding sites on Cys-loop LGICs. Stopped-flow fluorescence traces obtained upon mixing (Aalpha(2)-L1M/L38M)(2) with halothane revealed immediate, fast, and slow components of quenching. The immediate component, which occurred within the mixing time of the spectrofluorimeter, was attributed to direct quenching of tryptophan fluorescence upon halothane binding to (Aalpha(2)-L1M/L38M)(2). This was followed by a biexponential fluorescence decay containing fast and slow components, reflecting anesthetic-induced conformational transitions. Fluorescence traces obtained in studies using sevoflurane, isoflurane, and desflurane, which poorly quench tryptophan fluorescence, did not contain the immediate component. However, these anesthetics did produce the fast and slow components, indicating that they also alter the conformation of (Aalpha(2)-L1M/L38M)(2). Cyclopropane, an anesthetic that acts with unusually low potency on Cys-loop LGICs, acted with low apparent potency on (Aalpha(2)-L1M/L38M)(2). These results suggest that four-alpha-helix bundle proteins may be useful models of in vivo sites of action that allow the use of a wide range of techniques to better understand how anesthetic binding leads to changes in protein structure and function.

Anesthetics, Inhalation↗
PubMed2003

Design, synthesis, and evaluation of analogues of 3,3,3-trifluoro-2-hydroxy-2-phenyl-propionamide as orally available general anesthetics.

We have recently discovered a novel class of compounds that have oral general anesthetic activity, potent anticonvulsant activity, and minimal hemodynamic effects. The 3,3,3-trifluoro-2-hydroxy-2-phenyl-propionamide (1) demonstrated potent ability to reduce the minimum alveolar concentration (MAC) of isoflurane, with no effects on heart rate or blood pressure at therapeutic concentrations. Analogue 1 also had potent oral anticonvulsant activity against maximal electroshock (MES) and subcutaneous metrazol (scMET) models with a therapeutic index of 10 for MES activity. In this study, we further synthesized nine new racemic analogues and evaluated these compounds for effects on isoflurane MAC reduction and blood pressure. Preliminary data demonstrate potent reduction in the isoflurane MAC for two new compounds. Current mechanistic studies were unrevealing for effects on voltage-gated ion channels as a putative mechanism. Liposomal partitioning studies using (19)F NMR reveal that the aromatic region partitions into the core of the lipid. This partitioning correlated with general anesthetic activity of this class of compounds. Further, compound 1 was used at a concentration of 1 mM and slightly enhanced GABA(A) current in hippocampal neurons at 10 microM. Altogether, 3,3,3-trifluoro-2-hydroxy-2-phenyl-propionamide exhibited excellent oral general anesthetic activity and appears devoid of significant side effects (i.e., alterations in blood pressure or heart rate).

Administration, Oral↗
PubMed2006

Are target-family-privileged substructures truly privileged?

One of the early and effective approaches to G-coupled protein receptor target family library design was the analysis of a set of ligands for frequently occurring chemical moieties or substructures. Various methods ranging from frameworks analysis to pharmacophores have been employed to find these so-called target-family-privileged substructures. Although the use of these substructures is common practice in combinatorial library design and has produced leads, the methods used for finding them rarely verified their selectivity for the particular target family from which they were derived. The frequency of occurrence among ligands associated with a target receptor family is not a sufficient criterion for those substructures to receive the label of target-family-privileged substructure. This study explores the question of selectivity of ClassPharmer generated fragments for a series of target families: GPCRs, nuclear hormone receptors, serine proteases, protein kinases, and ligand-gated ion channels. In addition, a GPCR focused library and a random set of 10k compounds are examined in terms of their target-family-privileged-substructure composition. The results challenge the combinatorial chemistry concept of target-family-privileged substructures and suggest that many of these fragments may simply be drug-like or attractive for various receptors in accordance with the original definition of privileged substructures.

Combinatorial Chemistry Techniques↗
PubMed1992

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.

Animals↗
PubMed1994

Molecular and cellular mechanisms of general anaesthesia.

General anaesthetics are much more selective than is usually appreciated and may act by binding to only a small number of targets in the central nervous system. At surgical concentrations their principal effects are on ligand-gated (rather than voltage-gated) ion channels, with potentiation of postsynaptic inhibitory channel activity best fitting the pharmacological profile observed in general anaesthesia. Although the role of second messengers remains uncertain, it is now clear that anaesthetics act directly on proteins rather than on lipids.

Anesthesia, General↗
PubMed1999

Life without huntingtin: normal differentiation into functional neurons.

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmitter-gated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.

Animals↗
PubMed1996

An inhalational anesthetic binding domain in the nicotinic acetylcholine receptor.

To determine inhalational anesthetic binding domains on a ligand-gated ion channel, I used halothane direct photoaffinity labeling of the nicotinic acetylcholine receptor (nAChR) in native Torpedo membranes. [14C]Halothane photoaffinity labeling of both the native Torpedo membranes and the isolated nAChR was saturable, with Kd values within the clinically relevant range. All phospholipids were labeled, with greater than 95% of the label in the acyl chain region. Electrophoresis of labeled nAChR demonstrated no significant subunit selectivity for halothane incorporation. Within the alpha-subunit, greater than 90% of label was found in the endoprotease Glu-C digestion fragments which contain the four transmembrane regions, and the pattern was different from that reported for photoactivatable phospholipid binding to the nAChR. Unlabeled halothane reduced labeling more than did isoflurane, suggesting differences in the binding domains for inhalational anesthetics in the nAChR. These data suggest multiple similar binding domains for halothane in the transmembrane region of the nAChR.

Affinity Labels↗
PubMed1998

Oleamide potentiates benzodiazepine-sensitive gamma-aminobutyric acid receptor activity but does not alter minimum alveolar anesthetic concentration.

UNLABELLED: A naturally occurring brain lipid, cis-9,10-octadeceamide--oleamide (OA), is found in increased concentrations in the cerebrospinal fluid of sleep-deprived cats, which suggests that it may be an endogenous sleep-inducing substance. We studied the effects of this fatty-acid derivative on the function of cloned gamma-aminobutyric acid (GABA(A)) receptors expressed in Xenopus oocytes. Oocytes were injected with cRNA synthesized in vitro to express simple GABA(A) receptors (alpha1beta1, alpha3beta1, alpha5beta1, and alpha1beta2 subunit combinations) and receptors in which the GABA-induced chloride currents were potentiated in the presence of benzodiazepines (alpha1beta1gamma2s and alpha1beta2gamma2s subunit combinations). OA only produced significant potentiation of the peak Cl- current when applied with GABA to benzodiazepine-sensitive GABA(A) receptors. The peak currents of the simple GABA(A) receptors in the presence of OA were either unaffected or slightly inhibited by OA, but the overall mean currents were not significantly altered. Oleic acid was also capable of potentiating benzodiazepine-sensitive GABA(A) receptor function. The function of other ligand-gated ion channels, such as the N-methyl-D-aspartate receptor (NR1 + NR2A or 2C) and the 5-HT3 receptor expressed in Xenopus oocytes, were unaffected by OA. Sprague-Dawley rats receiving intraperitoneal injections of oleamide (10, 20, or 100 mg/kg) showed no change in the minimum alveolar anesthetic concentration (MAC) of desflurane required to abolish movement in response to noxious (tail clamp) stimulation (control MAC 6.48% +/- 1.28% atm; 100 mg/kg OA MAC 7.05% +/- 0.42% atm). These results reinforce the view that oleyl compounds may be natural modulators of inhibitory ion channel function, but that these effects contribute little to the central nervous system depression produced by volatile anesthetics as measured by MAC. IMPLICATIONS: The putative sleep-inducing substance, oleamide, potentiates benzodiazepine-sensitive gamma-aminobutyric acid receptor function but does not alter desflurane minimum alveolar anesthetic concentration in rats.

Anesthetics, Inhalation↗
PubMed1999

Electrical recordings from rat cardiac muscle cells using field-effect transistors.

Extracellular electrophysiological recordings were made from cardiac cells cultured for up to seven days over microfabricated arrays of field-effect transistors. The recorded signals can be separated mainly into two types of cell transistor couplings: one that can be explained entirely by purely passive circuitry elements, and a second where voltage-gated ion channels contribute greatly to the measured extracellular signal.

Action Potentials↗
PubMed2005

Modulation of human 5-hydroxytryptamine type 3AB receptors by volatile anesthetics and n-alcohols.

Functional 5-hydroxytryptamine type 3 (5-HT3) receptors can be formed by 5-HT3A subunits alone or in combination with the 5-HT3B subunit, but only the 5-HT3A receptor has been previously studied with respect to the modulation by volatile anesthetics and n-alcohols. Using two-electrode voltage-clamp, we show for the first time the modulation of heteromeric human (h)5-HT3AB receptors, expressed in Xenopus oocytes, by a series of n-alcohols and halogenated volatile anesthetics. At twice their anesthetic concentration, compounds having a molecular volume of less than 110 A3 enhanced submaximal 5-HT-evoked current. Compounds larger than 110 A3 inhibited submaximal 5-HT-evoked current. In experiments examining 5-HT concentration-response relationships, chloroform and butanol caused a slight decrease in the 5-HT EC50. Sevoflurane and octanol inhibited 5-HT-evoked current at all 5-HT concentrations tested but had no effect upon the 5-HT EC50. Compared with previous data on homomeric h5-HT3A receptors, the presence of the h5-HT3B subunit reduces the enhancement of h5-HT3 receptors by smaller halogenated volatile anesthetics and n-alcohols. In summary, these results suggest that heteromeric h5-HT3AB receptors are modulated by halogenated volatile anesthetics at clinically relevant concentrations, in addition to n-alcohols, suggesting that these receptors may be another physiological target for these compounds. The modulation is dependent upon the molecular volume of the compound, further supporting the concept of an anesthetic binding pocket of limited volume common on other Cys-loop ligand-gated ion channels. Incorporation of the 5-HT3B subunit alters either the anesthetic binding site or the allosteric interactions between anesthetic binding and channel opening.

Alcohols↗
PubMed2007

Life among the axons.

A blink in history's eye has brought us an understanding of electricity, and with it a revolution in human life. From the frog leg twitch experiments of Galvani and the batteries of Volta, we have progressed to telegraphs, motors, telephones, computers, and the Internet. In the same period, the ubiquitous role of electricity in animal and plant life has become clear. A great milestone in this journey was the elucidation of electrical signaling by Hodgkin & Huxley in 1952. This chapter gives a personal account of a small part of this story, the transformation of the rather abstract electrical conductances of Hodgkin & Huxley into the more tangible gated ion channel.

Animals↗
PubMed1999

Salivary gland P2 nucleotide receptors.

The effects of ATP on salivary glands have been recognized since 1982. Functional and pharmacological studies of the P2 nucleotide receptors that mediate the effects of ATP and other extracellular nucleotides have been supported by the cloning of receptor cDNAs, by the expression of the receptor proteins, and by the identification in salivary gland cells of multiple P2 receptor subtypes. Currently, there is evidence obtained from pharmacological and molecular biology approaches for the expression in salivary gland of two P2X ligand-gated ion channels, P2Z/P2X7 and P2X4, and two P2Y G protein-coupled receptors, P2Y1 and P2Y2. Activation of each of these receptor subtypes increases intracellular Ca2+, a second messenger with a key role in the regulation of salivary gland secretion. Through Ca2+ regulation and other mechanisms, P2 receptors appear to regulate salivary cell volume, ion and protein secretion, and increased permeability to small molecules that may be involved in cytotoxicity. Some localization of the various salivary P2 receptor subtypes to specific cells and membrane subdomains has been reported, along with evidence for the co-expression of multiple P2 receptor subtypes within specific salivary acinar or duct cells. However, additional studies in vivo and with intact organ preparations are required to define clearly the roles the various P2 receptor subtypes play in salivary gland physiology and pathology. Opportunities for eventual utilization of these receptors as pharmacotherapeutic targets in diseases involving salivary gland dysfunction appear promising.

Adenosine Triphosphate↗
PubMed1999

A zinc-dependent Cl- current in neuronal somata.

Extracellular Zn2+ modulates current passage through voltage- and neurotransmitter-gated ion channels, at concentrations less than, or near, those produced by release at certain synapses. Electrophysiological effects of cytoplasmic Zn2+ are less well understood, and effects have been observed at concentrations that are orders of magnitude greater than those found in resting and stimulated neurons. To examine whether and how neurons are affected by lower levels of cytoplasmic Zn2+, we tested the effect of Zn2+-selective chelators, Zn2+-preferring ionophores, and exogenous Zn2+ on neuronal somata during whole-cell patch-clamp recordings. We report here that cytoplasmic zinc facilitates the downward regulation of a background Cl- conductance by an endogenous protein kinase C (PKC) in fish retinal ganglion cell somata and that this regulation is maintained if nanomolar levels of free Zn2+ are available. This regulation has not been described previously in any tissue, as other Cl- currents have been described as reduced by PKC alone, reduced by Zn2+ alone, or reduced by both independently. Moreover, control of cation currents by a zinc-dependent PKC has not been reported previously. The regulation we have observed thus provides the first electrophysiological measurements consistent with biochemical measurements of zinc-dependent PKC activity in other systems. These results suggest that contributions of background Cl- conductances to electrical properties of neurons are susceptible to modulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗