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PubMed2005

Identification of the cyclic-nucleotide-binding domain as a conserved determinant of ion-channel cell-surface localization.

Mutations of a putative cyclic-nucleotide-binding domain (CNBD) can disrupt the function of the hyperpolarization-activated cyclic-nucleotide-gated channel (HCN2) and the human ether-a-go-go-related gene potassium channel (HERG). Loss of function caused by C-terminal truncation, which includes all or part of the CNBD in HCN and HERG, has been related to abnormal channel trafficking. Similar defects have been reported for several of the missense mutations of HERG associated with long QT syndrome type 2 (LQT2). Thus, we postulate that normal processing of these channels depends upon the presence of the CNBD. Here, we show that removal of the entire CNBD prevents Golgi transit, surface localization and function of HERG channel tetramers. This is also true when any of the structural motifs of the CNBD is deleted, suggesting that deletion of any highly conserved region along the entire length of the CNBD can disrupt channel trafficking. Furthermore, we demonstrate that defective trafficking is a consequence of all LQT2 mutations in the CNBD, including two mutations not previously assessed and two others for which there are conflicting results in the literature. The trafficking sensitivity of the CNBD might be of general significance for other ion channels because complete deletion of the CNBD or mutations at highly conserved residues within the CNBD of the related ERG3 channel and HCN2 also prevent Golgi transit. These results broadly implicate the CNBD in ion-channel trafficking that accounts for the commonly observed loss of function associated with CNBD mutants and provides a rationale for distinct genetic disorders.

Amino Acid Sequence↗
PubMed2000

Location of the polyamine binding site in the vestibule of the nicotinic acetylcholine receptor ion channel.

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, (125)I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies, we conclude that the site of (125)I-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of (125)I-MR44. The site where the hydrophobic head group of (125)I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with (125)I-MR44. We conclude that the polyamine moiety of (125)I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by (125)I-MR44 should therefore be accessible from the luminal side of the vestibule.

Binding Sites↗
PubMed2004

Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.

Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABA(A) receptor (GABA(A)R) ligand-binding domain predicts that beta2Glu155 is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the beta2Ile154-Asp163 region in GABA(A)R activation, each residue was individually mutated to cysteine and coexpressed with wild-type alpha1 subunits in Xenopus laevis oocytes. Seven mutations increased the GABA EC50 value (8- to 3400-fold), whereas three mutations (E155C, S156C, and G158C) also significantly increased the 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) K(I) value. GABA, SR-95531, and pentobarbital slowed N-biotinylaminoethyl methanethiosulfonate modification of T160C and D163C, indicating that beta2Thr160 and beta2Asp163 are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of beta2Glu155 resulted in spontaneously open GABA(A)Rs and differentially decreased the GABA, piperidine-4-sulfonic acid (partial agonist), and SR-95531 sensitivities, indicating that the mutation perturbs ligand binding as well as channel gating. Tethering thiol-reactive groups onto beta2E155C closed the spontaneously open channels, suggesting that beta2Glu155 is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the beta2 Ile154-Asp163 region is a protein hinge that forms a network of interconnections that couples binding site movements to the cascade of events leading to channel opening.

Animals↗
PubMed2001

Cyclic-nucleotide-gated channels: pore topology in desensitizing E19A mutants.

In cyclic-nucleotide-gated "CNG" channels, the pore-loop "P-loop" is formed by the amino acid residues R345-S371 (here called R1-S27). Residue E19 determines the channel's interaction with extracellular divalent cations and contributes to ion conduction. Neutralization of this residue with alanine introduces channel desensitization. We have used serial cysteine mutagenesis to study P-loop topology in the alpha subunit of the mammalian rod CNG channels containing the E19A substitution. The pore topology was tested in the closed channel state and, when cGMP was present, during and after desensitization. With E19A substitution, the T15C, T16C, I17C and T20C mutants desensitized more slowly than controls. Moreover, the typical rundown produced by the I17C substitution in the wild-type "w.t." background was considerably reduced. Overall, with the E19A substitution, the accessibility pattern tested by applying the thiol-specific reagents Cd2+ and MTSET from the cytoplasmic side of the plasma membrane was similar to that observed with the w.t. Moreover, P22C channels were not inhibited by Cd2+ and MTSET (which do not cross the lipid bilayer) applied from the inside, but were blocked by MTSEA (which permeates the plasma membrane) also applied from the inside. This suggests that the residues following E19 remain accessible from the external side after E19A substitution. Thus, although the residues T15 to T20 seemed to participate in the structural rearrangements producing desensitization, no major P-loop remodelling occurs in desensitizing channels.

Amino Acid Sequence↗
PubMed2003

Protons and calcium alter gating of the hyperpolarization-activated cation current (I(h)) in rod photoreceptors.

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I(h), in rod photoreceptors. I(h) is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I(h) activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca(2+)] from 3 to 20 mM resulted in a large (approximately 17 mV) positive shift in I(h) activation. External [Ca(2+)] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (-9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I(h) activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I(h) activation.

Ambystoma↗
PubMed1997

unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C. elegans locomotion.

Mechanically gated ion channels are important modulators of coordinated movement, yet little is known of their molecular properties. We report that C. elegans unc-8, originally identified by gain-of-function mutations that induce neuronal swelling and severe uncoordination, encodes a DEG/ENaC family member homologous to subunits of a candidate mechanically gated ion channel. unc-8 is expressed in several sensory neurons, interneurons, and motor neurons. unc-8 null mutants exhibit previously unrecognized but striking defects in the amplitude and wavelength of sinusoidal tracks inscribed as they move through an E. coli lawn. We hypothesize that UNC-8 channels could modulate coordinated movement in response to body stretch. del-1, a second DEG/ENaC family member coexpressed with unc-8 in a subset of motor neurons, might also participate in a channel that contributes to nematode proprioception.

Amino Acid Sequence↗
PubMed1998

Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization.

Although ATP-gated ion channel (P2XR) expression is high among anterior pituitary cells, identification of the receptor subtypes and their selective expression within subpopulations of cell types, as well as their physiological role(s), are incompletely characterized. In this study, we focused on the expression and activity of the P2X2R subtype in anterior pituitary cells. Our results indicate that the primary P2X2R gene transcript in pituitary cells undergoes extensive alternative splicing, with generation of six isoforms. Two of these isoforms encode functional channels when expressed in GT1 or HEK293 cells: the wild-type P2X2R and the spliced isoform P2X2-2R, which lacks a stretch of carboxyl-terminal amino acids (Val370-Gln438). Four other clones showed different alterations, including an interfered reading frame starting in the first transmembrane domain and a 27-amino acid deletion in the large extracellular loop. When expressed separately or in combination with wild-type channels, these clones were nonfunctional. In single cell Ca2+ current and cytosolic Ca2+ concentration ([Ca2+)i) measurements, the P2X2R and P2X2-2R had similar EC50 values for ATP and time courses for activation and recovery from desensitization but differed significantly in their desensitization rates. The spliced isoform exhibited rapid and complete desensitization, whereas the wild-type channel desensitized slowly and incompletely. The mRNAs for wild-type and spliced channels were identified in enriched somatotroph, but not gonadotroph or lactotroph fractions. Expression of a functional ATP-gated channel in somatotrophs was confirmed by the ability of ATP to increase the frequency of [Ca2+]i spikes in spontaneously active cells or initiate spiking in quiescent cells. When voltage-gated Ca2+ influx was blocked, ATP increased [Ca2+]i, with a similar profile and EC50 to those observed in GT1 cells heterologously expressing wild-type or spliced P2X2R. The ligand-selectivity profile of native channels was consistent with the presence of P2X2R in somatotrophs. Finally, the desensitization rate of P2X2R in a majority of somatotrophs was comparable to that observed in neurons coexpressing wild-type and spliced channels. These data indicate that alternative splicing of P2X2R and coexpression of P2X2R and P2X2-2R subunits provide effective mechanisms for controlled cationic influx in somatotrophs.

Adenosine Triphosphate↗
PubMed1995

The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating.

A highly conserved leucine is found in the middle of the porelining (M2) domain of the members of the ligand-gated ion channel family. Two very different roles have been proposed for this leucine. In one model, this residue swings into the lumen of the channel during desensitization to form the nonconducting desensitized state, whereas in the other model, the leucines from each subunit interact with each other to form a constriction in the channel that constitutes the actual gate of the channel. We examined the role of this leucine in the muscle-type acetylcholine receptor by replacing it with the polar amino acid threonine. Replacement of the leucine in any one subunit slows desensitization and shifts the dose-response relationship toward lower concentrations. Replacement of leucines in additional subunits leads to progressively larger shifts in the dose-response curves. The shift depends only on the number of leucines replaced, not on which particular subunits contain the mutation; in other words, the mutations act independently. At the single-channel level, the mutation greatly increases the channel mean open time. We conclude that the role of the conserved leucine is to set the mean open time of the channel through interactions with other regions of the receptor rather than to serve as the gate per se of the ion channel.

Animals↗
PubMed2002

Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity.

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective alpha1 homomeric glycine receptor (alpha1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1'E GlyR) was sufficient to convert the channels to select cations over anions with P(Cl)/P(Na) = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2'Delta mutant GlyR retained its anion selectivity (P(Cl)/P(Na) = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (P(Cl)/P(Na) = 27.9). When the A-1'E and the P-2'Delta mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (P(Cl)/P(Na) = 0.13). The SDM GlyR was also Ca(2+) permeable (P(Ca)/P(Na) = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more Ca(2+)-permeable channel (P(Ca)/P(Na) = 0.73), without drastically altering monovalent charge selectivity (P(Cl)/P(Na) = 0.23). The SDM+R19'E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca(2+) permeability (P(Ca)/P(Na) = 0.92), with little effect on monovalent selectivity (P(Cl)/P(Na) = 0.19). Estimates of the minimum pore diameter of the A-1'E, SDM, SDM+R19'A, and SDM+R19'E GlyRs revealed that these pores are larger than the alpha1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.

Amino Acid Sequence↗
PubMed2000

In vivo structure-function analyses of Caenorhabditis elegans MEC-4, a candidate mechanosensory ion channel subunit.

Mechanosensory signaling mediated by mechanically gated ion channels constitutes the basis for the senses of touch and hearing and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. We report a genetically based structure-function analysis of the candidate mechanotransducing channel subunit MEC-4, a core component of a touch-sensing complex in Caenorhabditis elegans and a member of the DEG/ENaC superfamily. We identify molecular lesions in 40 EMS-induced mec-4 alleles and further probe residue and domain function using site-directed approaches. Our analysis highlights residues and subdomains critical for MEC-4 activity and suggests possible roles of these in channel assembly and/or function. We describe a class of substitutions that disrupt normal channel activity in touch transduction but remain permissive for neurotoxic channel hyperactivation, and we show that expression of an N-terminal MEC-4 fragment interferes with in vivo channel function. These data advance working models for the MEC-4 mechanotransducing channel and identify residues, unique to MEC-4 or the MEC-4 degenerin subfamily, that might be specifically required for mechanotransducing function. Because many other substitutions identified by our study affect residues conserved within the DEG/ENaC channel superfamily, this work also provides a broad view of structure-function relations in the superfamily as a whole. Because the C. elegans genome encodes representatives of a large number of eukaryotic channel classes, we suggest that similar genetic-based structure-activity studies might be generally applied to generate insight into the in vivo function of diverse channel types.

Amino Acid Sequence↗
PubMed2003

How fast does the GluR1Qflip channel open?

Opening of a ligand-gated ion channel is the step at which the binding of a neurotransmitter is transduced into the electrical signal by allowing ions to flow through the transmembrane channel, thereby altering the postsynaptic membrane potential. We report the kinetics for the opening of the GluR1Qflip channel, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit of the ionotropic glutamate receptors. Using a laser-pulse photolysis technique that permits glutamate to be liberated photolytically from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (caged glutamate) with a time constant of approximately 30 micros, we show that, after the binding of glutamate, the channel opened with a rate constant of (2.9 +/- 0.2) x 10(4) s(-1) and closed with a rate constant of (2.1 +/- 0.1) x 10(3) s(-1). The observed shortest rise time (20-80% of the receptor current response), i.e. the fastest time by which the GluR1Qflip channel can open, was predicted to be 35 micros. This value is three times shorter than those previously reported. The minimal kinetic mechanism for channel opening consists of binding of two glutamate molecules, with the channel-opening probability being 0.93 +/- 0.10. These findings identify GluR1Qflip as one of the temporally efficient receptors that transduce the binding of chemical signals (i.e. glutamate) into an electrical impulse.

Cell Line↗
PubMed1992

The size of gating charge in wild-type and mutant Shaker potassium channels.

The high sensitivity of voltage-gated ion channels to changes in membrane potential implies that the process of channel opening is accompanied by large charge movements. Previous estimates of the total charge displacement, q, have been deduced from the voltage dependence of channel activation and have ranged from 4 to 8 elementary charges (e0). A more direct measurement of q in Drosophila melanogaster Shaker 29-4 potassium channels yields a q value of 12.3 e0. A similar q value is obtained from mutated Shaker channels having reduced voltage sensitivity. These results can be explained by a model for channel activation in which the equilibria of voltage-dependent steps are altered in the mutant channels.

Animals↗
PubMed2003

EXP-1 is an excitatory GABA-gated cation channel.

Gamma-aminobutyric acid (GABA) mediates fast inhibitory neurotransmission by activating anion-selective ligand-gated ion channels. Although electrophysiological studies indicate that GABA may activate cation-selective ligand-gated ion channels in some cell types, such a channel has never been characterized at the molecular level. Here we show that GABA mediates enteric muscle contraction in the nematode Caenorhabditis elegans via the EXP-1 receptor, a cation-selective ligand-gated ion channel. The EXP-1 protein resembles ionotropic GABA receptor subunits in almost all domains. In the pore-forming domain of EXP-1, however, the residues that confer anion selectivity are exchanged for those that specify cation selectivity. When expressed in Xenopus laevis oocytes, EXP-1 forms a GABA receptor that is permeable to cations and not anions. We conclude that some of the excitatory functions assigned to GABA are mediated by cation channels rather than by anion channels.

Amino Acid Sequence↗
PubMed2005

Structural basis of gating of CNG channels.

Cyclic nucleotide-gated (CNG) ion channels, underlying sensory transduction in vertebrate photoreceptors and olfactory sensory neurons, require cyclic nucleotides to open. Here, we present structural models of the tetrameric CNG channel pore from bovine rod in both open and closed states, as obtained by combining homology modeling-based techniques, experimentally derived spatial constraints and structural patterns present in the PDB database. Gating is initiated by an anticlockwise rotation of the N-terminal region of the C-linker, which is then, transmitted through the S6 transmembrane helices to the P-helix, and in turn from this to the pore lumen, which opens up from 2 to 5A thus allowing for ion permeation. The approach, here presented, is expected to provide a general methodology for model ion channels and their gating when structural templates are available and an extensive electrophysiological analysis has been performed.

Amino Acid Sequence↗
PubMed2003

Movements near the gate of a hyperpolarization-activated cation channel.

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Like the related depolarization-activated K+ channels (Kv channels), HCN channels use an intracellular activation gate to regulate access to an inner cavity, lined by the S6 transmembrane regions, which leads to the selectivity filter near the extracellular surface. Here we describe two types of metal interactions with substituted cysteines in the S6, which alter the voltage-controlled movements of the gate. At one position (L466), substitution of cysteine in all four subunits allows Cd2+ ions at nanomolar concentration to stabilize the open state (a "lock-open" effect). This effect depends on native histidines at a nearby position (H462); the lock-open effect can be abolished by changing the histidines to tyrosines, or enhanced by changing them to cysteines. Unlike a similar effect in Kv channels, this effect depends on a Cd2+ bridge between 462 and 466 in the same subunit. Cysteine substitution at another position (Q468) produces two effects of Cd2+: both a lock-open effect and a dramatic slowing of channel activation-a "lock-closed" effect. The two effects can be separated, because the lock-open effect depends on the histidine at position 462. The novel lock-closed effect results from stabilization of the closed state by the binding of up to four Cd2+ ions. During the opening conformational change, the S6 apparently moves from one position in which the 468C cysteines can bind four Cd2+ ions, possibly as a cluster of cysteines and cadmium ions near the central axis of the pore, to another position (or flexible range of positions) where either 466C or 468C can bind Cd2+ in association with the histidine at 462.

Amino Acid Sequence↗
PubMed2006

Sequence-dependent gating of an ion channel by DNA hairpin molecules.

DNA hairpins produce ionic current signatures when captured by the alpha-hemolysin nano-scale pore under conditions of single molecule electrophoresis. Gating patterns produced by individual DNA hairpins when captured can be used to distinguish differences of a single base pair or even a single nucleotide [Vercoutere,W.A. et al. (2003) Nucleic Acids Res., 31, 1311-1318]. Here we investigate the mechanism(s) that may account for the ionic current gating signatures. The ionic current resistance profile of conductance states produced by DNA hairpin molecules with 3-12 bp stems showed a plateau in resistance between 10 and 12 bp, suggesting that hairpins with 10-12 bp stems span the pore vestibule. DNA hairpins with 9-12 bp stems produced gating signatures with the same relative conductance states. Systematic comparison of the conductance state dwell times and apparent activation energies for a series of 9-10 bp DNA hairpins suggest that the 3' and 5' ends interact at or near the limiting aperture within the vestibule of the alpha-hemolysin pore. The model presented may be useful in predicting and interpreting DNA detection using nanopore detectors. In addition, this well-defined molecular system may prove useful for investigating models of ligand-gated channels in biological membranes.

Bacterial Toxins↗
PubMed2006

Receptor-independent effects of endocannabinoids on ion channels.

Endogenous cannabinoids (endocannabinoids), produced from membrane-bound precursors via calcium and/or G-protein dependent processes, mimic the effects of cannabinoids by activating cannabinoid CB(1) and/or CB(2) receptors. Several reports however, also indicate that endocannabinoids can produce effects that are independent of cannabinoid receptors. Thus, in pharmacologically relevant concentrations, endocannabinoids have been demonstrated to modulate the functional properties of voltage-gated ion channels including Ca(2+) channels, Na(+) channels and various types of K(+) channels, and ligand-gated ion channels such as 5-HT(3), and nicotinic ACh receptors. In addition, the functional modulations by endocannabinoids of other ion-transporting membrane proteins such as transient potential receptor-class channels, gap junctions, and neurotransmitter transporters have also been reported. These findings indicate that additional molecular targets for endocannabinoids exist and that these targets may represent important sites for cannabinoids to alter either the excitability of the neurons or the response of the neuronal systems. This review focuses on the results of recent studies indicating that beyond their receptor-mediated effects, endocannabinoids alter the function of ion channels directly.

Animals↗