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PubMed2004

Techniques: Use of concatenated subunits for the study of ligand-gated ion channels.

Members of the pentameric ligand-gated ion channel family, comprising GABA(A) receptors, nicotinic acetylcholine receptors, glycine receptors and 5-HT3 receptors, are involved in information transfer at both synapses and the neuromuscular junction. However, receptors that are composed of five subunits are difficult to analyse by recombinant expression of a mixture of the single subunits because multiple receptor subtypes with different subunit composition or arrangement can be formed. Covalently linking the C-terminus of the preceding subunit with the N-terminus of the following subunit to form a concatenated subunit enables the precise predetermination of subunit arrangement in these receptors. A forced subunit assembly enables the characterization of: (i) receptor architecture; (ii) properties of receptors that contain different subunit isoforms in specific locations; and (iii) selective introduction of a mutation into a specific subunit that occurs multiple times in a receptor. Thus, this method also facilitates the investigation of positional effects of mutations associated with diseases.

Animals↗
PubMed2002

Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels.

In cyclic nucleotide-gated (CNG) ion channels, binding of cGMP or cAMP drives a conformational change that leads to opening of an ion-conducting pore. One region implicated in the coupling of ligand binding to opening of the pore is the C linker region. Here, we used crosslinking of endogenous cysteines to study interregion proximity. We demonstrate that an individual amino acid--C481--in the C linker region of each of two neighboring subunits can form a disulfide bond. Further, using tandem dimers, we show that a disulfide bond between C35 in the N-terminal region and C481 in the C linker region can form either within a subunit or between subunits. From our data on proximity between individual amino acids and previous studies, a picture emerges of the C linker as a potential dimerization interface.

Animals↗
PubMed2004

2-aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3.

Six of the mammalian transient receptor potential (TRP) ion channel subtypes are nonselective cation channels that can be activated by increases or decreases in ambient temperature. Five of them can alternatively be activated by nonthermal stimuli such as capsaicin [transient receptor potential vanilloid 1 (TRPV1)] or hypo-osmolarity (TRPV2 and TRPV4). No nonthermal stimuli have yet been described for TRPV3, a warmth-gated ion channel expressed prominently in skin keratinocytes. Here, we demonstrate that 2-aminoethoxydiphenyl borate (2-APB), a compound used to inhibit store-operated Ca2+ channels and IP3 receptors, produces robust activation of recombinant TRPV3 in human embryonic kidney 293 cells with an EC50 of 28 microm. 2-APB also sensitizes TRPV3 to activation by heat, even at subthreshold concentrations. In inside-out membrane patches from TRPV3-expressing cells, 2-APB increases the open probability of TRPV3. Also, whereas heat alone is capable of activating TRPV3-mediated currents in only a small proportion of primary mouse keratinocytes, 2-APB activates heat-evoked, TRPV3-mediated currents in the majority of these cells. Together, these findings identify 2-APB as the first known chemical activator of TRPV3 and enhance the notion that TRPV3 participates in the detection of heat by keratinocytes.

Animals↗
PubMed1989

Markov, fractal, diffusion, and related models of ion channel gating. A comparison with experimental data from two ion channels.

The gating kinetics of single-ion channels are generally modeled in terms of Markov processes with relatively small numbers of channel states. More recently, fractal (Liebovitch et al. 1987. Math. Biosci. 84:37-68) and diffusion (Millhauser et al. 1988. Proc. Natl. Acad. Sci. USA. 85:1502-1507) models of channel gating have been proposed. These models propose the existence of many similar conformational substrates of the channel protein, all of which contribute to the observed gating kinetics. It is important to determine whether or not Markov models provide the most accurate description of channel kinetics if progress is to be made in understanding the molecular events of channel gating. In this study six alternative classes of gating model are tested against experimental single-channel data. The single-channel data employed are from (a) delayed rectifier K+ channels of NG 108-15 cells and (b) locust muscle glutamate receptor channels. The models tested are (a) Markov, (b) fractal, (c) one-dimensional diffusion, (d) three-dimensional diffusion, (e) stretched exponential, and (f) expo-exponential. The models are compared by fitting the predicted distributions of channel open and closed times to those observed experimentally. The models are ranked in order of goodness-of-fit using a boot-strap resampling procedure. The results suggest that Markov models provide a markedly better description of the observed open and closed time distributions for both types of channel. This provides justification for the continued use of Markov models to explore channel gating mechanisms.

Animals↗
PubMed1994

Using sequence homology to analyze the structure and function of voltage-gated ion channel proteins.

Molecular modeling and mutagenesis analysis of voltage-gated channels have succeeded in identifying much of the topology of the proteins and in identifying which sequential segments are involved in functional mechanisms such as activation gating, inactivation gating, ion selectivity, and ligand binding. Efforts are currently underway to use these methods to model the protein structure and functional mechanisms more precisely. The experimental and theoretical efforts are dependent to a considerable extent upon information obtained by comparing homologous sequences. Although the fine details of models developed in this manner are unlikely to be as correct as models developed from x-ray crystallography and NMR, they still may contribute substantially to our understanding of the structure and function of these important proteins.

Amino Acid Sequence↗
PubMed2003

Cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels were first discovered in rod photoreceptors, where they are responsible for the primary electrical signal of the photoreceptor in response to light. CNG channels are highly specialized membrane proteins that open an ion-permeable pore across the membrane in response to the direct binding of intracellular cyclic nucleotides. CNG channels have been identified in a number of other tissues, including the brain, where their roles are only beginning to be appreciated. Recently, significant progress has been made in understanding the molecular mechanisms underlying their functional specializations. From these studies, a picture is beginning to emerge for how the binding of cyclic nucleotide is transduced into the opening of the pore and how this allosteric transition is modulated by various physiological effectors.

Allosteric Regulation↗
PubMed2003

Ion channel gating: insights via molecular simulations.

Ion channels are gated, i.e. they can switch conformation between a closed and an open state. Molecular dynamics simulations may be used to study the conformational dynamics of ion channels and of simple channel models. Simulations on model nanopores reveal that a narrow (<4 A) hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small (approximately 1 A) increase in pore radius or an increase in polarity. Modelling and simulation studies confirm the importance of hydrophobic gating in K channels, and support a model in which hinge-bending of the pore-lining M2 (or S6 in Kv channels) helices underlies channel gating. Simulations of a simple outer membrane protein, OmpA, indicate that a gate may also be formed by interactions of charged side chains within a pore, as is also the case in ClC channels.

Bacterial Outer Membrane Proteins↗
PubMed1997

Evolutionary relationship of the ligand-gated ion channels and the avermectin-sensitive, glutamate-gated chloride channels.

Two cDNAs, GluClalpha and GluClbeta, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707-711, 1994). Expression studies in Xenopus oocytes showed that GluClalpha and GluClbeta have pharmacological profiles distinct from the glutamate-gated cation channels as well as the gamma-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClalpha and GluClbeta genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClalpha and GluClbeta form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClalpha and GluClbeta, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClalpha and GluClbeta belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels.

Animals↗
PubMed2001

Proline-induced hinges in transmembrane helices: possible roles in ion channel gating.

A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.

Alamethicin↗
PubMed1994

Single 5-HT3 receptor-gated ion channel events resolved in N1E-115 mouse neuroblastoma cells.

In order to resolve single channel events underlying the 5-HT3 receptor-gated ion current in N1E-115 neuroblastoma cells patch clamp experiments have been performed on excised outside-out membrane patches under optimized experimental conditions. When the driving force for Na+ through the 5-HT3 receptor-gated ion channel is enhanced by raising the external Na+ concentration to 180 mM and by substituting internal K+ with the relatively impermeable cation N-methyl-D-glucamine, a single conductance level is observed in the presence of 5-HT. Single channel activity is observed only in the presence of the agonist and is blocked by 50 nM of the selective 5-HT3 receptor antagonist MDL 72222 in a reversible manner. At membrane potentials more negative than -60 mV discrete single channel events can be resolved with a conductance of 5.6 +/- 1.2 pS.

Animals↗
PubMed2006

The cys-loop ligand-gated ion channel superfamily of the honeybee, Apis mellifera.

Members of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily mediate neurotransmission in insects and are targets of successful insecticides. We have described the cys-loop LGIC superfamily of the honeybee, Apis mellifera, which is an important crop pollinator and a key model for social interaction. The honeybee superfamily consists of 21 genes, which is slightly smaller than that of Drosophila melanogaster comprising 23 genes. As with Drosophila, the honeybee possesses ion channels gated by acetylcholine, gamma-amino butyric acid, glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel (pHCl), CG8916, CG12344 and CG6927. Similar to Drosophila, honeybee cys-loop LGIC diversity is broadened by differential splicing which may also serve to generate species-specific receptor isoforms. These findings on Apis mellifera enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.

Alternative Splicing↗
PubMed1997

Alcohols and neurotransmitter gated ion channels: past, present and future.

The neurotransmitter-gated ion channels form a superfamily of neurotransmitter receptors specialized for recognizing transmitters and rapidly gating ion channels that are contained within the same holoprotein complex. A large body of research indicates that alcohols alter the function of this class of receptor at concentrations relevant to the intoxicating and anesthetic effects of the alcohols. In addition, studies have implicated several types of neurotransmitter-gated channels in intoxicating and anesthetic alcohol actions. All of the neurotransmitter-gated ion channels contain conserved features such as N-terminal ligand binding domains, hydrophobic membrane spanning domains and charged pore-lining domains. However, at least two, and possibly three families of receptors have been identified within the superfamily, including the nicotinic ACh-like receptors, the ionotropic glutamate receptors and the ATP-gated ion channels. This review will begin with a brief overview of the structural features of the different receptors, with an emphasis on comparing and contrasting features of the different families of neurotransmitter-gated channels. The emphasis will be mostly on the nicotinic-like receptors and the iGluRs, since more is known about these receptors than about other types of ligand-activated cation channels. The remainder of the review focuses on the latest studies aimed at determining the mechanism of alcohol actions on this superfamily of receptor-channels as well as the relationship between the molecular structure of these channels and the effects of alcohols on channel function. In addition, emerging directions for future study of these effects of alcohols and possible regions of the protein that may be altered during alcohol exposure are discussed.

Animals↗
PubMed1995

Unraveling the modular design of glutamate-gated ion channels.

Glutamate receptors that function as ligand-gated ion channels are essential components of cell-cell communication in the nervous system. Despite a wealth of information concerning these receptors, details of their structure are just beginning to emerge. We propose that glutamate receptors comprise four modules: two modules that are related to bacterial periplasmic-binding proteins, one module that is related to the pore-forming region of K+ channels, and one regulatory module of unknown origin. A K(+)-channel-like domain inserted into a crucial region of a periplasmic-binding protein-like domain suggests a mechanism for transduction of binding energy to channel opening. This modular design also suggests an evolutionary link between a ligand-gated ion-channel family and voltage-gated ion channels.

Amino Acid Sequence↗
PubMed1995

Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.

Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs--a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology.

Alkaline Phosphatase↗
PubMed2004

Fractional diffusion modeling of ion channel gating.

An anomalous diffusion model for ion channel gating is put forward. This scheme is able to describe nonexponential, power-law-like distributions of residence time intervals in several types of ion channels. Our method presents a generalization of the discrete diffusion model by Millhauser, Salpeter, and Oswald [Proc. Natl. Acad. Sci. U.S.A. 85, 1503 (1988)] to the case of a continuous, anomalous slow conformational diffusion. The corresponding generalization is derived from a continuous-time random walk composed of nearest-neighbor jumps which in the scaling limit results in a fractional diffusion equation. The studied model contains three parameters only: the mean residence time, a characteristic time of conformational diffusion, and the index of subdiffusion. A tractable analytical expression for the characteristic function of the residence time distribution is obtained. In the limiting case of normal diffusion, our prior findings [Proc. Natl. Acad. Sci. U.S.A. 99, 3552 (2002)] are reproduced. Depending on the chosen parameters, the fractional diffusion model exhibits a very rich behavior of the residence time distribution with different characteristic time regimes. Moreover, the corresponding autocorrelation function of conductance fluctuations displays nontrivial power law features. Our theoretical model is in good agreement with experimental data for large conductance potassium ion channels.

Animals↗
PubMed2003

International Union of Pharmacology. XL. Compendium of voltage-gated ion channels: calcium channels.

This summary article presents an overview of the molecular relationships among the voltage-gated calcium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the calcium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Calcium Channel Blockers↗
PubMed1998

Alcohol effects on the 5-HT3 ligand-gated ion channel.

1. Alcohols and volatile anesthetics have both potentiating and channel blocking effects on members of the nicotinic ACh-like subfamily of ligand-gated ion channels. 2. The alcohol and anesthetic sensitivity of the 5-HT3 receptor, a member of this receptor subfamily, has been examined extensively by several laboratories. 3. Alcohols and volatile anesthetics potentiate receptor function. 4. This potentiation is characterized by an increase in the apparent potency with which agonists activate the receptor, and an apparent stabilization and favoring of the open channel state. 5. Long-chain alcohols and trichloroethanol also inhibit channel function, perhaps through a channel blocking mechanism similar to that seen at the nACh receptor. 6. Studies of the relationship between receptor structure and alcohol and anesthetic effects are just beginning, but some clues from other members of this subfamily of ligand-gated channels may help to pinpoint areas of the receptor that are important for alcohol effects.

Anesthetics↗