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PubMed1982

A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5-A resolution.

The crystal structure of alamethicin in nonaqueous solvent has been determined, and refined at 1.5-A resolution. The molecular conformation of the three crystallographically independent molecules is largely alpha-helical with a bend in the helix axis at an internal proline residue. The helix structure is highly amphipathic as most of the solvent-accessible polar atoms lie on a narrow strip of surface parallel to the helix axis. Molecular models for the voltage-gated ion channel, with n-fold symmetry and based on the molecular conformations observed in the crystal, are characterized by strong surface complementarity, a hydrophilic interior and a hydrophobic exterior. The channel structures are stabilized by a hydrated annulus of hydrogen-bonded glutamine residues which produce the greatest restriction in the channel diameter.

Alamethicin↗
PubMed1990

A radiohistochemical measure of [3H]TCP binding to the activated NMDA-receptor-gated ion channel in rat brain.

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor is linked to an ion channel that is blocked by the phencyclidine analog N-(1-[thienyl]cyclohexyl)piperidine (TCP). Previous studies have shown that NMDA and glycine act together to increase the access of [3H]TCP to its binding site, presumably by increasing channel opening; NMDA/glycine-enhanced [3H]TCP binding performed under non-equilibrium conditions thereby serves as a dynamic molecular marker of channel activation. In this study we tested whether NMDA and glycine regulate [3H]TCP binding in slide-mounted brain sections. Striking activation of the NMDA ion channel was observed in neo- and allocortex; dentate gyrus and strata radiatum and oriens of CA1 and CA3 of hippocampal formation; and certain amygdaloid nuclei (basomedial, basolateral, and cortical). Other nuclei of the amygdala (central, posterolateral, and medial), basal ganglia, and numerous regions within the diencephalon, brainstem and cerebellum showed relatively little activation of the NMDA ion channel. Our demonstration of enriched NMDA/glycine-stimulated [3H]TCP binding in stratum radiatum of hippocampal region CA1 but not in cerebellar granule cell layer correlates with electrophysiologic studies that showed NMDA channel ion flux in CA1 but not in cerebellar granule cell layer in adult rats. These data demonstrate that NMDA and glycine regulation of [3H]TCP binding can be quantified with a radiohistochemical method to provide a regional measure of the activated NMDA-receptor-gated ion channel. This technique is a powerful tool for functional analysis of the NMDA receptor/channel complex in both physiologic and pathologic states.

Animals↗
PubMed1990

Developmental and tissue-specific expression of the rod photoreceptor cGMP-gated ion channel gene.

Probes against the retinal cGMP-gated cation channel were generated by PCR amplification of cDNA from rat and bovine retina. Southern and Northern analyses showed that the channel is encoded by a single gene that gives rise to a single mRNA species of 3.2 kb. Low levels of cGMP-gated channel RNA were detected in postnatal day 1 (PN 1) retinas and the amount increased to adult levels over the next two weeks of development. Screening of a number of tissues by Northern blot hybridization and by PCR amplification showed the channel to be expressed by heart and kidney as well as retina, but not by cerebellum, cerebral cortex, liver, muscle, olfactory bulb, spleen, testes or thymus.

Aging↗
PubMed1996

Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels.

Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular loop. Functionally, P2X5 and P2X6 receptors most resemble P2X2 and P2X4; they desensitize only slowly and do not respond to alpha beta methylene-ATP. P2X6 receptors, like P2X4, receptors, are not blocked by the antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. P2X6 and P2X5 receptors express at lower levels than P2X1-P2X4 receptors do, perhaps indicating that they do not normally form homomultimeric channels. P2X6 and P2X4 are the receptors expressed most heavily in brain, where their RNAs have a widespread and extensively overlapping distribution. The spinal cord expresses all receptors except P2X3. P2X2, P2X4, and P2X6, are the most abundant in the dorsal horn. Sensory neurons of the trigeminal, dorsal root, and nodose ganglia express all six RNAs; P2X3 is found only there. The functional properties and tissue distribution of these six P2X receptors indicate new roles for ATP-gated ion channels.

Adenosine Triphosphate↗
PubMed2001

Structure of the pore-forming transmembrane domain of a ligand-gated ion channel.

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.

Animals↗
PubMed1998

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

UNLABELLED: P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. KEYWORDS: blue native PAGE/cross-linking/P2X receptor/quaternary structure.

Animals↗
PubMed2004

A quantitative assessment of models for voltage-dependent gating of ion channels.

Voltage-gated ion channels open and close, or "gate," in response to changes in membrane potential. The electric field across the membrane-protein complex exerts forces on charged residues driving the channel into different functional conformations as the membrane potential changes. To act with the greatest sensitivity, charged residues must be positioned at key locations within or near the transmembrane region, which requires desolvating charged groups, a process that can be energetically prohibitive. Although there is good agreement on which residues are involved in this process for voltage-activated potassium channels, several different models of the sensor geometry and gating motions have been proposed. Here we incorporate low-resolution structural information about the channel into a Poisson-Boltzmann calculation to determine solvation barrier energies and gating charge values associated with each model. The principal voltage-sensing helix, S4, is represented explicitly, whereas all other regions are represented as featureless, dielectric media with complex boundaries. From our calculations, we conclude that a pure rotation of the S4 segment within the voltage sensor is incapable of producing the observed gating charge values, although this shortcoming can be partially remedied by first tipping and then minimally translating the S4 helix. Models in which the S4 segment has substantial interaction with the low-dielectric environment of the membrane incur solvation energies of hundreds of k(B)T, and activation times based on these energies are orders of magnitude slower than experimentally observed.

Amino Acid Sequence↗
PubMed2004

Arabidopsis DND2, a second cyclic nucleotide-gated ion channel gene for which mutation causes the "defense, no death" phenotype.

A previous mutant screen identified Arabidopsis dnd1 and dnd2 "defense, no death" mutants, which exhibit loss of hypersensitive response (HR) cell death without loss of gene-for-gene resistance. The dnd1 phenotype is caused by mutation of the gene encoding cyclic nucleotide-gated (CNG) ion channel AtCNGC2. This study characterizes dnd2 plants. Even in the presence of high titers of Pseudomonas syringae expressing avrRpt2, most leaf mesophyll cells in the dnd2 mutant exhibited no HR. These plants retained strong RPS2-, RPM1-, or RPS4-mediated restriction of P. syringae pathogen growth. Mutant dnd2 plants also exhibited enhanced broad-spectrum resistance against virulent P. syringae and constitutively elevated levels of salicylic acid, and pathogenesis-related (PR) gene expression. Unlike the wild type, dnd2 plants responding to virulent and avirulent P. syringae exhibited elevated expression of both salicylate-dependent PR-1 and jasmonate and ethylene-dependent PDF1.2. Introduction of nahG+ (salicylate hydroxylase) into the dnd2 background, which removes salicylic acid and causes other defense alterations, eliminated constitutive disease resistance and PR gene expression but only weakly impacted the HR- phenotype. Map-based cloning revealed that dnd2 phenotypes are caused by mutation of a second CNG ion channel gene, AtCNGC4. Hence, loss of either of two functionally nonredundant CNG ion channels can cause dnd phenotypes. The dnd mutants provide a unique genetic background for dissection of defense signaling.

Arabidopsis↗
PubMed1995

Spontaneous, ligand-independent activity of the cGMP-gated ion channels in cone photoreceptors of fish.

1. We studied the electrical conductance of membrane patches detached from the outer segment of single cone photoreceptors isolated from striped bass retina. 2. Only a single class of ion channels exists in the plasma membrane of the cone outer segments; they are gated by cytoplasmic cGMP and select cations over anions, but distinguish poorly among cations. In the absence of added cGMP and of divalent cations, however, membrane patches detached from the outer segments exhibit a small conductance that ideally selects cations over anions, but distinguishes poorly between Na+ and Li+. 3. The cGMP-independent conductance does not arise from the effect of residual cGMP that may remain associated with the detached membrane, because treatment of the patch with cGMP-specific phosphodiesterase does not affect this conductance. 4. The cGMP-independent conductance is pharmacologically indistinguishable from that activated by cGMP. Ca2+ and L-cis-diltiazem block both conductances at comparable concentrations and with similar quantitative characteristics. 5. We analysed the noise of Ca(2+)- or L-cis-diltiazem-dependent macroscopic currents both in the presence and in the absence of cGMP. In the presence of cGMP, the power density spectrum of the noise is well fitted by the sum of two Lorentzian components. The same function with similar corner frequencies fits the noise of the cGMP-independent currents. However, the total power in the current fluctuations is smaller in the absence of cGMP than in its presence; also, the ratio of the zero frequency asymptotes of the low over the high frequency components, S1(0)/Sh(0), is larger in the absence of cGMP than in its presence.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure.

The diameters of small arteries and arterioles are tightly regulated by the dynamic interaction between Ca(2+) and K(+) channels in the vascular smooth muscle cells. Calcium influx through voltage-gated Ca(2+) channels induces vasoconstriction, whereas the opening of K(+) channels mediates hyperpolarization, inactivation of voltage-gated Ca(2+) channels, and vasodilation. Three types of voltage-sensitive ion channels have been highly implicated in the regulation of resting vascular tone. These include the L-type Ca(2+) (Ca(L)) channels, voltage-gated K(+) (K(V)) channels, and high-conductance voltage- and Ca(2+)-sensitive K(+) (BK(Ca)) channels. Recently, abnormal expression profiles of these ion channels have been identified as part of the pathogenesis of arterial hypertension and other vasospastic diseases. An increasing number of studies suggest that high blood pressure may trigger cellular signaling cascades that dynamically alter the expression profile of arterial ion channels to further modify vascular tone. This article will briefly review the properties of Ca(L), K(V), and BK(Ca) channels, present evidence that their expression profile is altered during systemic hypertension, and suggest potential mechanisms by which the signal of elevated blood pressure may result in altered ion channel expression. A final section will discuss emerging concepts and opportunities for the development of new vasoactive drugs, which may rely on targeting disease-specific changes in ion channel expression as a mechanism to lower vascular tone during hypertensive diseases.

Arteries↗
PubMed1997

Point mutations in alpha bENaC regulate channel gating, ion selectivity, and sensitivity to amiloride.

We have generated two site-directed mutants, K504E and K515E, in the alpha subunit of an amiloride-sensitive bovine epithelial Na+ channel, alpha bENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E alpha bENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (Ki) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E alpha bENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as being important to the regulation of fundamental channel properties.

Amiloride↗
PubMed1996

GABAc receptors: relatively simple transmitter -gated ion channels?

The inhibitory neurotransmitter, GABA, activates a variety of receptors in all areas of the CNS. Two major subtypes of GABA receptors are well known: (1) GABAA receptors are ligand-gated Cl- channels that consist of a heteromeric mixture of protein subunits forming a pentameric structure, and (2) GABAB receptors couple to Ca2+ and K+ channels via G proteins and second messengers. Here, Graham Johnston discusses evidence for a third major subclass of GABA receptors. GABAC receptors appear to be relatively simple ligand-gated Cl- channels with a distinctive pharmacology, in that they are not blocked by bicuculline and not modulated by barbiturates, benzodiazepines or neuroactive steroids. Compared with GABAA receptors, GABAC receptors are activated at lower concentrations of GABA and are less liable to desensitization. In addition, their channels open for a longer time. The pharmacology of these novel subtypes of GABA receptors may yield important therapeutic agents.

Animals↗
PubMed1999

Expression of two ATP-gated ion channels, P2X5 and P2X6, in developing chick skeletal muscle.

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand-gated ion-channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle.

Amino Acid Sequence↗