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PubMed1992

Electromagnetic gating in ion channels.

There have been many attempts to develop a theoretical explanation of the phenomena of electromagnetic field interactions with biological systems. None of the reported efforts have been entirely successful in accounting for the observed experimental results, in particular with respect to the reports of interactions between extremely low frequency (ELF) magnetic fields and biological systems at ion cyclotron resonance frequencies. The approach used in this paper starts with the Lorentz force equation, but use is made of cylindrical co-ordinates and cylindrical boundary conditions in an attempt to more closely model the walls of an ion channel. The equations of motion of an ion that result from this approach suggest that the inside shape of the channel plus the ELF magnetic fields at specific frequencies and amplitudes could act as a gate to control the movement of the ion across the cell membrane.

Animals↗
PubMed2000

Roles of ion channels in EPSP integration at neuronal dendrites.

Many different kinds of voltage-gated ion channels (Na+ channels, K+ channels, Ca2+ channels) exist at neuronal dendrites. Integration of dendritic electric signals (excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs) and action potentials) and/or non-electric signals (Ca2+ and second messengers) occurs in restricted dendritic compartments consisting of spines and adjacent fine dendrites. Voltage-gated ion channels at neuronal dendrites play crucial roles in the integration of dendritic signals. Dendritic signals, in turn, play important roles in the modulation of local dendritic physiological functions (e.g. input-specific synaptic plasticity, long-term potentiation (LTP) and long term depression (LTD)). A combined experimental and theoretical approach is a good way to clarify the biophysical behaviors of dendritic ion channels. Analyses of dendritic ion channels can open the door to a new wave of discoveries about EPSP integration at neuronal dendrites.

Animals↗
PubMed2003

Fluorescence imaging of electrically stimulated cells.

Designing high-throughput screens for voltage-gated ion channels has been a tremendous challenge for the pharmaceutical industry because channel activity is dependent on the transmembrane voltage gradient, a stimulus unlike ligand binding to G-protein-coupled receptors or ligand-gated ion channels. To achieve an acceptable throughput, assays to screen for voltage-gated ion channel modulators that are employed today rely on pharmacological intervention to activate these channels. These interventions can introduce artifacts. Ideally, a high-throughput screen should not compromise physiological relevance. Hence, a more appropriate method would activate voltage-gated ion channels by altering plasma membrane potential directly, via electrical stimulation, while simultaneously recording the operation of the channel in populations of cells. The authors present preliminary results obtained from a device that is designed to supply precise and reproducible electrical stimuli to populations of cells. Changes in voltage-gated ion channel activity were monitored using a digital fluorescent microscope. The prototype electric field stimulation (EFS) device provided real-time analysis of cellular responsiveness to physiological and pharmacological stimuli. Voltage stimuli applied to SK-N-SH neuroblastoma cells cultured on the EFS device evoked membrane potential changes that were dependent on activation of voltage-gated sodium channels. Data obtained using digital fluorescence microscopy suggests suitability of this system for HTS.

Calcium↗
PubMedDate not supplied

Generation of a focused set of GSK compounds biased toward ligand-gated ion-channel ligands.

A "data mining" methodology based on substructural analysis and standard 1024 Daylight fingerprints as descriptors was applied to a set of known antagonists of a subfamily of ligand-gated ion channels comprising nicotinic acetylcholine receptors (nAChR's), 5-hydroxytryptamine, gamma-amino butyric acid-A, and glycine receptors. The derived scoring function was used to generate a focused set that was screened for alpha7 nAChR, resulting in the identification of novel alpha7 ligands easily amenable to chemical modification. Finally, the same scoring function was applied retrospectively to other in-house sets screened for the same target in the same assay. The results and performance of the method are described in detail.

Algorithms↗
PubMed2000

Cyclic nucleotide-gated channels in non-sensory organs.

Cyclic nucleotide-gated channels represent a class of ion channels activated directly by the binding of either cyclic-GMP or cyclic-AMP. They carry both mono and divalent cations, but select calcium over sodium. In the majority of the cases studied, binding of cyclic nucleotides to the channel results in the opening of the channel and the influx of calcium. As a consequence, cytosolic free calcium levels increase leading to the modifications of calcium-dependent processes. This represents and important link in the chain of events leading to the physiological response. Cyclic nucleotide-gated channels were discovered in sensory cell types, in the retina, and in olfactory cells, and were extensively studied in those cells. However, it is becoming increasingly evident that such channels are present not only in sensory systems, but in most, if not all, cell types where cyclic nucleotides play a role in signal transduction. A hypothesis is presented here which attributes physiological importance to these channels in non-sensory organs. Four examples of such channels in non-sensory cells are discussed in detail: those in the liver, in the heart, in the brain, and in the testis with the emphasis on the possible physiological roles that these channels might have in these organs.

Animals↗
PubMed1992

GABAA receptors: ligand-gated Cl- ion channels modulated by multiple drug-binding sites.

GABAA receptors are ligand-gated Cl- ion channels and the site of action of a variety of pharmacologically and clinically important drugs. In this review evidence is summarized indicating that these drugs, by interacting with several distinct binding sites at these receptors, allosterically modulate GABA-induced Cl- ion flux. Other results indicate that the affinity, as well as the modulatory efficacy of drugs, changes with receptor composition. A though investigation of the pharmacological properties of the individual binding sites on different GABAA receptor subtypes could open new avenues for selective modulation of GABAA receptors in different brain regions.

Animals↗
PubMed2003

Molecular basis of the effect of potassium on heterologously expressed pacemaker (HCN) channels.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels modulate the firing rates of neuronal and cardiac pacemaker cells. HCN channels resemble voltage-gated K+ channels structurally, but much less is known about their structure-function correlation. Although modulation of K+ channel gating by external K+ is a well-known phenomenon, such a link has not been established for HCN channels. Here we examined the effects of external permeant (K+, Na+ and Li+) and non-permeant (NMG+) ions on HCN1 and HCN2 gating. Substituting 64 of 96 mM external K+ with Na+, Li+ or NMG+ positively shifted steady-state activation (approximately 13 mV), and preferentially slowed activation of HCN1. Mutating the pore variant C-terminal to the GYG motif in HCN1, A352, to the analogous conserved Asp in K+ channels or Arg in HCN2 produced a significant hyperpolarizing activation shift (by 5-15 mV), slowed gating kinetics (up to 6-fold), and abolished or attenuated gating responses to external K+. Whereas Na+, Li+ and NMG+ substitutions produced depolarizing activation shifts of HCN2 similar to those of HCN1, deactivation but not activation of HCN2 was exclusively decelerated. We conclude that gating and permeation of HCN channels are coupled, and that modulation of this 'pore-to-gate' coupling by external K+ is isoform-specific.

Amino Acid Motifs↗
PubMed2000

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels.

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.

Animals↗
PubMed2003

A multiply charged tetracaine derivative blocks cyclic nucleotide-gated channels at subnanomolar concentrations.

Cyclic nucleotide-gated (CNG) ion channels are central participants in sensory transduction, generating the electrical response to light in retinal photoreceptors and to odorants in olfactory receptors. They are expressed in many other tissues where their specific roles in signaling remain unclear. As is true for many other ion channels, there is a paucity of specific blockers needed to dissect the contributions of these channels to cell signaling. CNG channels are members of the superfamily of voltage-gated ion channels, and the local anesthetic tetracaine is known to block CNG channels in a manner that resembles the block of voltage-gated Na(+) channels. The amine in local anesthetics interacts with the charged selectivity filter of Na(+) channels, while the aromatic ring gets stuck in the inner cavity and has hydrophobic interactions with the residues lining that region. Here we have synthesized a derivative of tetracaine, 3-[(aminopropyl)amino]-N,N-dimethyl-N-(2-[[4-(butylamino)benzoyl]oxy]ethyl)propan-1-aminium acetate (APPA-tetracaine), that contains three positively charged amines at physiological pH instead of one. This compound blocked several different CNG channels in the picomolar to nanomolar concentration range at positive membrane potentials, making it several orders of magnitude more potent than tetracaine. In contrast, significant block of Na(+) channels by APPA-tetracaine required concentrations of hundreds of nanomolar. The results suggest that the highly charged moiety of APPA-tetracaine interacts strongly with the negative charge cluster in the selectivity filter of CNG channels. We propose that a variety of potent and specific ion channel blockers could be generated by expanding on traditional blocker structures to target the selectivity filters of other channels.

Animals↗
PubMed1996

An amino acid substitution in the pore region of a glutamate-gated chloride channel enables the coupling of ligand binding to channel gating.

Many of the subunits of ligand-gated ion channels respond poorly, if at all, when expressed as homomeric channels in Xenopus oocytes. This lack of a ligand response has been thought to result from poor surface expression, poor assembly, or lack of an agonist binding domain. The Caenorhabditis elegans glutamate-gated chloride channel subunit GluClbeta responds to glutamate as a homomeric channel while the GluClalpha subunit is insensitive. A chimera between GluClalpha and GluClbeta was used to suggest that major determinants for glutamate binding are present on the GluClalpha N terminus. Amino acid substitutions in the presumed pore of GluClalpha conferred direct glutamate gating indicating that GluClalpha is deficient in coupling of ligand binding to channel gating. Heteromeric channels of GluClalpha+beta may differ from the prototypic muscle nicotinic acetylcholine receptor in that they have the potential to bind ligand to all of the subunits forming the channel.

Amino Acid Sequence↗
PubMed2002

Movement of the C-helix during the gating of cyclic nucleotide-gated channels.

Movements within the cyclic nucleotide-binding domain of cyclic nucleotide-gated channels are thought to underlie the initial phase of channel gating (Tibbs, G. R., D. T. Liu, B. G. Leypold, and S. A. Siegelbaum. 1998. J. Biol. Chem. 273:4497-4505; Zong, X., H. Zucker, F. Hofmann, and M. Biel. 1998. EMBO J. 17:353-362; Matulef, K., G. E. Flynn, and W. N. Zagotta. 1999. Neuron. 24:443-452; Paoletti, P., E. C. Young, and S. A. Siegelbaum. 1999. J. Gen. Physiol. 113:17-33; Johnson, J. P., and W. N. Zagotta. 2001. Nature. 412:917-921). To investigate these movements, cysteine mutation was performed on each of the 28 residues (Leu-583 to Asn-610), which span the agonist-binding domain of the alpha-subunit of the bovine rod cyclic nucleotide-gated channel. The effects of Cd(2+) ions, 2-trimethylammonioethylmethane thiosulfonate (MTSET) and copper phenanthroline (CuP) on channel activity were examined, in excised inside-out patches in the presence and in the absence of a saturating concentration of cGMP. The application of 100 microM Cd(2+) in the presence of saturating concentration of cGMP caused an irreversible and almost complete reduction of the current in mutant channels E594C, I600C, and L601C. In the absence of cGMP, the presence of 100 microM Cd(2+) caused a strong current reduction in all cysteine mutants from Asp-588 to Leu-607, with the exception of mutant channels A589C, M592C, M602C, K603C, and L606C. The selective effect of Cd(2+) ions was very similar to that observed when adding the oxidizing agent CuP to the bath medium, except for mutant channel G597C, where CuP caused a stronger current decrease (67 +/- 7%) than Cd(2+) (23 +/- 4%). In the absence of cGMP, MTSET caused a reduction of the current by >40% in mutant channels L607C, L601C, I600C, G597C, and E594C, whereas in the presence of cGMP only mutant channel L601C was affected. The application of MTSET protected many mutant channels from the effects of Cd(2+) and CuP. These results suggest that, when CNG channels are in the open state, residues from Asp-588 to Leu-607 are in an alpha-helical structure, homologous to the C-helix of the catabolite gene activator protein (Weber, I. T., and T. A. Steitz. 1987. J. Mol. Biol. 198:311-326). Furthermore, residues Glu-594, Gly-597, Ile-600, and Leu-601 of these helices belonging to two different subunits must be in close proximity. In the closed state the C-helices are in a different configuration and undergo significant fluctuations.

Amino Acid Sequence↗
PubMed2005

Pore-to-gate coupling of HCN channels revealed by a pore variant that contributes to gating but not permeation.

Although ample evidence suggests the presence of an intracellular activation gate in HCN (pacemaker) channels, mutations in the outer pore can alter gating properties. Here we investigated the role of the outer pore residue A354 in HCN1 gating by systematically converting it to the equivalent residues (T, Y, and F) found in K(+)-channels. A354T negatively shifted steady-state activation (DeltaV(1/2) approximately -25 mV), decelerated gating kinetics (by up to 8-fold), and abolished the effects of external ions on gating. A354Y and A354F did not yield functional currents when expressed alone, although immunofluorescence microscopy indicated the presence of these channel proteins on the membrane surface. Currents recorded after co-expressing A354Y with WT HCN1 were reduced in amplitude (relative to WT alone) and had changes in gating similar to those of A354T. We conclude that the pore variant at position 354 contributes to gating but not permeation, and that the HCN outer pore may be involved in gating via a pore-to-gate coupling mechanism.

Alanine↗
PubMed2004

Function and structure in glycine receptors and some of their relatives.

In the field of ligand-gated ion channels, recent developments, both in the knowledge of structure and in the measurement of function at the single-channel level, have allowed a sensible start to be made on understanding the relationship between structure and function in these proteins. In this review, the cases of glycine, nicotinic ACh and glutamate receptors are compared and contrasted, and problems such as how binding of agonist causes the channel to open, and why partial agonists are partial, are considered. Some observations, both structural and functional, suggest that more attention needs to be paid to conformational changes that occur before the channel opens. Such changes might account for the interaction found between subunits of the glycine receptor while it is still shut and, perhaps, the agonist-dependent structural changes seen in AMPA receptors. They might also complicate our understanding of the binding-gating problem.

Animals↗
PubMed1999

Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy.

Voltage-gated ion channels are transmembrane proteins that are essential for nerve impulses and regulate ion flow across cell membranes in response to changes in membrane potential. They are made up of four homologous domains or subunits, each of which contains six transmembrane segments. Studies of potassium channels have shown that the second (S2) and fourth (S4) segments contain several charged residues, which sense changes in voltage and form part of the voltage sensor. Although these regions clearly undergo conformational changes in response to voltage, little is known about the nature of these changes because voltage-dependent distance changes have not been measured. Here we use lanthanide-based resonance energy transfer to measure distances between Shaker potassium channel subunits at specific residues. Voltage-dependent distance changes of up to 3.2 A were measured at several sites near the S4 segment. These movements directly correlated with electrical measurements of the voltage sensor, establishing the link between physical changes and electrical charge movement. Measured distance changes suggest that the region associated with the S4 segment undergoes a rotation and possible tilt, rather than a large transmembrane movement, in response to voltage. These results demonstrate the first in situ measurement of atomic scale movement in a trans-membrane protein.

Cysteine↗
PubMed2003

Molecular coupling between voltage sensor and pore opening in the Arabidopsis inward rectifier K+ channel KAT1.

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.

Algorithms↗
PubMed2005

A mathematical model of ion movements in grey matter during a stroke.

The development of cytotoxic oedema during a stroke consists in cell swelling and shrinking of the extracellular space. This phenomenon is triggered by ion movements through voltage-gated channels, exchangers and pumps. During ischaemia, sodium, calcium and chloride enter the neurons whereas potassium and glutamate are expelled out of the cells. A mathematical model is proposed to represent the long-term dynamics of membrane potentials, cell volumes and ionic concentrations in intracellular and extracellular spaces during a stroke and to study the influence of each ionic current on cell swelling. The model relies on electrophysiological mechanisms and takes into account the behaviour of two types of cells: neurons and also astrocytes known to play a key role in the excitotoxic process in grey matter. The results obtained when a severe or a moderate ischaemia is simulated are consistent with those observed in the in vitro and in vivo experiments. As this model appears to be robust, it is used to perform illustrative simulations aimed at studying the effect of some channel blockers on cell swelling. This approach may help to explore new therapeutic strategies in order to reduce stroke damage.

Astrocytes↗
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↗