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At least 667 records · Page 37Linked to original sources
PubMed1988

Inhibition of the GABA receptor-gated chloride ion channel in brain by noncompetitive inhibitors of the nicotinic receptor-gated cation channel.

The ability of various noncompetitive inhibitors (NCI) of the nicotinic acetylcholine receptor to inhibit gamma-aminobutyric acidA (GABA) receptor activity in brain was investigated. Micromolar concentrations of NCI such as tetraphenylphosphonium, mepacrine, chlorpromazine and phencyclidine inhibited muscimol-induced 36chloride (36Cl-) uptake in rat cerebral cortical synaptoneurosomes in a noncompetitive manner. D-Tubocurarine behaved as a competitive inhibitor. In experiments measuring the time course of muscimol-induced 36Cl- uptake, a decline in the apparent transport rate constant (k') (reflection of desensitization) occurred over the first 3 sec. At lowered temperature (22 degrees C) the k' did not decline during the first 4 sec. Under these conditions preincubation of the vesicles with tetraphenylphosphonium caused a marked decline in the apparent k' over the first 5 sec (transition T1/2 of 1.04 sec) suggesting that tetraphenylphosphonium promotes desensitization of the GABA receptor. The inhibition of muscimol-induced 36Cl- uptake by NCI was reduced in the absence of Ca++. In addition, Ca++ decreased the potency of muscimol to stimulate 36Cl- uptake with an IC50 = 64 microM. The interaction of NCI with GABA agonist and convulsant sites associated with the GABA receptor-gated Cl- channel also was investigated. NCI antagonized noncompetitively [3H]t-butylbicycloorthobenzoate binding to convulsant sites whereas only mepacrine and chlorpromazine antagonized binding of [3H]muscimol to agonist sites. These findings suggest that 1) inhibition of the GABA receptor-gated Cl- channel by NCI may explain the convulsant properties of several of these compounds and 2) there may be structural domains common to the GABA receptor and nicotinic receptor-gated ion channels that selectively permit interactions with various NCI.

Animals↗
PubMed2004

A model of the closed form of the nicotinic acetylcholine receptor m2 channel pore.

The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.

Cell Membrane↗
PubMed1993

Stratification of the channel domain in neurotransmitter receptors.

Analyses of the ionic pore of ligand-gated ion channels at the amino acid level reveal a structural and functional stratification of the M2 channel domain. Mutations in the equatorial and outer regions affect channel gating, whereas mutations of other amino acid rings alter ionic permeability or selectivity.

Animals↗
PubMed1997

Molecular approaches to receptors as targets for drug discovery.

The cloning of a great number of receptors and channels has revealed that many of these targets for drug discovery can be grouped into superfamilies based on sequence and structural similarities. This review presents an overview of how molecular biological approaches have revealed a plethora of receptor subtypes, led to new definitions of subtypes and isoforms, and played a role in the development of high selective drugs. Moreover, the diversity of subtypes has molded current views of the structure and function of receptor families. Practical difficulties and limitations inherent in the characterization of the ligand binding and signaling properties of expressed recombinant receptors are discussed. The importance of evaluating drug-receptor interactions that differ with temporally transient and distinct receptor conformational states is emphasized. Structural motifs and signal transduction features are presented for the following major receptor superfamilies: ligand-gated ion channel, voltage-dependent ion channel, G-protein coupled, receptor tyrosine-kinase, receptor protein tyrosine-phosphatase, cytokine and nuclear hormone. In addition, a prototypic receptor is analyzed to illustrate functional properties of a given family. The review concludes with a discussion of future directions in receptor research that will impact drug discovery, with a specific focus on orphan receptors as targets for drug discovery. Methods for classifying orphan receptors based upon homologies with members of existing superfamilies are presented together with molecular approaches to the greater challenge of defining their physiological roles. Besides revealing new orphan receptors, the human genome sequencing project will result in the identification of an abundance of novel receptors that will be molecular targets for the development of highly selective drugs. These findings will spur the discovery and development of an exciting new generation of receptor-subtype specific drugs with enhanced therapeutic specificity.

Amino Acid Sequence↗
PubMed2000

Paradoxical effects of mineralocorticoids on the ion gated sodium channel in embryologically diverse cells.

PCR analysis and Western blotting revealed the expression of the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes at the level of RNA, DNA, and protein in several leukemic cell lines, fibroblasts from human cornea, and epithelial cells from ocular tissues. Following immunofluorescence, the MCR appeared to be primarily nuclear whereas the ENaC was almost exclusively membrane-bound. Paradoxically, the MCR-specific antagonist ZK 91587 actually stimulated the multiplication of human erythroblastic leukemia cells, contrary to the inhibitory effect of the antagonist RU 26752 on the multiplication of corneal fibroblasts; both effects were opposed by aldosterone. In quantitative PCR, both basal and aldosterone-induced levels of ENaC were diminished by ZK 91587 in the corneal fibroblast, in contrast to the stimulation observed in the retinal pigmentary epithelium. Thus, contrary to the existing notions, (a) antimineralocorticoids can act both as agonists and antagonists, and (b) the receptor-mediated action of mineralocorticoids on the sodium channel is not restricted to the epithelial cell.

Aldosterone↗
PubMed2003

Voltage-gated K channels.

Ion channels and the electrical properties they confer on cells are involved in every human characteristic that distinguishes us from the stones in a field. Every perception, thought, movement, and heartbeat depends on electrical signals generated by the activity of ion channels. Early views of the relationship between channel structure and function have undergone substantial modification following the cloning of various ion channels and the determination of the structure of a simple bacterial K channel, the KcsA channel. This review focuses on the relationship between the structure and function of voltage-dependent K channels, covering the molecular bases of channel selectivity, conduction, and gating. The evolution of ion channels in bacteria is discussed, as well as the basis of channel selectivity and conduction in the KcsA channel. More complex channels have evolved molecular "gatekeepers," allowing them to respond to appropriate stimuli by opening, closing, and inactivating.

Animals↗
PubMed2000

Stretch-activated signaling of nerve growth factor secretion in bladder and vascular smooth muscle cells from hypertensive and hyperactive rats.

Elevated vascular (VSMC) and bladder smooth muscle (BSMC) NGF are associated with altered visceral innervation in the spontaneously hypertensive rat (SHR: hypertensive, behaviorally hyperactive) compared with control Wistar-Kyotos (WKYs). Stretch stimulates increased NGF production in BSMCs. To elucidate whether stretch induces NGF synthesis in VSMCs, and to determine if disturbances in stretch-mediated NGF production contribute to the elevated tissue levels of NGF in SHRs, we subjected VSMCs and BSMCs cultured from four established inbred rat strains (WKY, WKHA: hyperactive; SHR and WKHT: hypertensive) to several stretch paradigms. For VSMCs, acute and cyclic stretch affected cells derived from hypertensive rats (80-100% increase over control) but not from normotensive strains. For BSMCs, cyclic and static stretch increased NGF secretion in all four strains, but had a two- to threefold greater effect in cells from SHRs and WKHTs (increase up to 600%) at early time points. At later time points of a 24-h experimental period, stretch increased NGF output up to 400% in SHR and WKHA cultures. Thus, defects that influence early induction of stretch-mediated SHR NGF secretion cosegregate with the hypertensive phenotype. Stretch-gated ion channel inhibitors, voltage-gated ion channel inhibitors, and protease inhibitors failed to affect stretch-induced BSMC NGF secretion. In contrast, gene transcription, intracellular calcium, protein kinase C (PKC), and autocrine release of an unknown factor may play a role in the elevated NGF secretion observed in smooth muscle from hypertensive animals. Altered stretch-induced smooth muscle NGF secretion may contribute to the augmented vascular and bladder NGF content associated with high blood pressure and hyperactive voiding in SHRs.

Animals↗
PubMed2005

Functional significance of HCN2/3-mediated I(h) in striatal cells at early developmental stages.

Hyperpolarization-activated cAMP-gated cation currents (I(h)) were recently linked to pre- and postnatal developmental processes in several brain regions, including the ventral telencephalon. To evaluate the role of I(h) in striatal development, we used short-term cultured cells from the lateral ganglionic eminence at embryonic day 14 (E14) and postnatal days 1-3 (P1-3) as well as the embryonic striatal progenitor cell line ST14A. Western blot analysis of the I(h) underlying subunit proteins HCN1-4 revealed strong HCN2 expression in proliferating ST14A cells and weak expression in postmitotic ST14A cells and in cells from the developing brain. We also found HCN3 expression only in ST14A cells at both proliferative and nonproliferative stages but not in short-term cultured striatal cells. In all cases, HCN1 and HCN4 transcripts were below the detection level. Despite the selective protein expression, RT-PCR analysis showed stable expression of HCN2-4 but not HCN1 mRNA in all short-term-cultured striatal cells and in the ST14A cell line. Consistent with the strong protein expression, an I(h) was recorded with features of an HCN2-mediated current in ST14A cells at the proliferative stage and in short-term-cultured E14 cells. Of particular importance is that we detected no currents upon hyperpolarization in the ST14A cells at the nonproliferative stage when only HCN3 protein was present. These results suggest the potential importance of ST14A cells in defining the molecular mechanisms regulating I(h) expression and function.

Animals↗
PubMed1992

A possible biological role of the electron transfer between tyrosine and tryptophan. Gating of ion channels.

Experiments have demonstrated that four tryptophan residues are located near the tetrodotoxin binding site in Na+ channels, and that conserved tyrosine and tryptophan residues are located in the pore-forming region of voltage-sensitive K+ channels. This paper proposes an activation mechanism involving electron transfer between these residues. The K+ channel may be closed by four tyrosine residues forming hydrogen bonds with each other. After electron transfer, these hydrogen bonds will be broken, thereby opening the channel. The Na+ channel could be activated by a similar mechanism. This idea can be tested directly by observing tyrosine or tryptophan radicals when the channels are in the open state.

Electron Transport↗
PubMed1995

Cyclic nucleotide-gated channels in visual and olfactory transduction.

Rod and cone photoreceptors are the light detectors in the visual system whereas olfactory receptor cells are the odorant detectors in the olfactory system. Despite the two very different types of stimuli, light in photoreceptors, and odorant molecules in olfactory receptor cells, the mechanisms of visual and olfactory transduction appear to have many homologies. Both stimuli trigger a chain of enzymatic events that terminates in a change in the concentration of a cyclic nucleotide: a decrease in the concentration of cGMP in photoreceptors, and an increase in the concentration of cAMP in olfactory receptor cells. These cyclic nucleotides directly gate cation channels and therefore a change in their concentration induced by the external stimulus is converted into an electrical signal. The analysis of the ionic selectivity properties of cyclic nucleotidegated channels in retinal rods, cones and in olfactory receptor cells shows that there are many similarities between these channels. They do not appreciably select between alkali monovalent cations and can be permeated and blocked by divalent cations. Their ionic permeation properties are consistent with the presence of a cation-binding site of high-field strength in the pore.

Animals↗
PubMed1995

Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels.

Sensory transduction in vertebrate photoreceptors and olfactory sensory neurons is mediated by cyclic nucleotide-gated (CNG) channels that conduct mono- and divalent cations. Ca2+ entering the cell through CNG channels intimately controls signaling pathways by regulating several key enzymes. Cloned CNG channels from photoreceptors and olfactory sensory neurons profoundly differ in their relative Ca2+ permeability, their blockage by external divalent cations, and the fraction of current carried by Ca2+. In particular, CNG channels from cone photoreceptors conduct significantly more Ca2+ than those from rod photoreceptors. Furthermore, the current through the olfactory CNG channel is entirely carried by Ca2+ at approximately 3 mM extracellular Ca2+. These results suggest that a major function of CNG channels is to provide a pathway for Ca2+ entry.

Animals↗
PubMed2006

Organisation of the mouse sinoatrial node: structure and expression of HCN channels.

OBJECTIVE: To reveal the structural characteristics of the sinoatrial node (SAN) and the distribution of hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN) in the SAN in the mouse. METHODS: The structure of the SAN and the distribution of HCN channels in the SAN in the mouse were studied by histology and immunolabelling of ANP, Cx43 and HCN channels. RESULTS: The mouse SAN is a comma-shaped structure with a length of approximately 1.5 mm parallel to the crista terminalis and is separated from atrial muscle by connective tissue at the border both with the crista terminalis and the atrial septum. A unique compact nodal structure with densely-packed nodal cells was identified at the head of the comma-shaped SAN. Cell size and fibre orientation vary regionally in the SAN: the cells in the compact node are small and are orientated perpendicular to the crista terminalis, whereas the cells in the more inferior part are larger and more loosely-packed and are orientated parallel to the crista terminalis. All SAN cells exhibited labelling of HCN4, but no cell exhibited detectable labelling of HCN1, HCN2, ANP and Cx43, while surrounding atrial cells exhibited labelling of ANP and Cx43, but not HCN1, HCN2 and HCN4. A specialised interface between the SAN and surrounding atrial muscle was also identified: strands of HCN4-positive nodal cells protrude into the atrial muscle and strands of Cx43-positive atrial cells protrude into the SAN; thus, there are interdigitations between the SAN and atrial muscle. CONCLUSIONS: In the mouse, (i) the SAN is structurally complex with a densely-packed head and loosely-packed tail; (ii) HCN4 is the only HCN isoform detectable and is present throughout the SAN; and (iii) there is a specialised interface between the SAN and surrounding atrium that may be necessary for the SAN to drive the more hyperpolarized atrial muscle.

Action Potentials↗
PubMed2002

Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated, cyclic nucleotide-gated channels.

In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms. To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy. We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo- or heteromeric channels on the plasma membrane of Chinese hamster ovary cells. We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels. This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein. However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel. Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays. Thus, interaction between NH(2)-terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels.

Animals↗
PubMed2004

Structural elements of instantaneous and slow gating in hyperpolarization-activated cyclic nucleotide-gated channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits produce a slowly activating current in response to hyperpolarization (If) and an instantaneous voltage-independent current (Iinst) when expressed in Chinese hamster ovary (CHO) cells. Here we found that a mutation in the S4-S5 linker of HCN2 (Y331D) produced an additional mixed cationic instantaneous current. However, this current was inhibited by external Cs+ like If and unlike Iinst. Together with a concomitant reduction in If, the data suggest that the Y331D mutation disrupted channel closing placing the channel in a "If-like," and not an "Iinst-like," state. The "If-like" instantaneous current represented approximately 70% of total If over voltages ranging from +20 to -150 mV in high K+ solutions. If activated at more depolarized potentials and the activation curve was less steep, whereas deactivation was significantly slowed, consistent with the idea that the mutation inhibited channel closing. The data suggest that the mutation produced allosteric effects on the activation gate (S6 segment) and/or on voltage-sensing elements. We also found that decreases in the ratio of external K+/Na+ further disrupted channel closing in the mutant channel. Finally, our data suggest that the structures involved in producing Iinst are similar between the HCN1 and HCN2 isoforms and that excess HCN protein on the plasma membrane of CHO cells relative to native cells is not responsible for Iinst. The data are consistent with Iinst flowing through a "leaky" closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits.

Amino Acid Sequence↗