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PubMed1983

Gating of ion channels made by a diphtheria toxin fragment in phospholipid bilayer membranes.

B45, a fragment containing the major hydrophobic region of diphtheria toxin, increases the conductance of thin lipid membranes by forming ion-conducting channels that are gated by transmembrane voltage, Vm, and the bath pH. Single-channel currents show "bursting" behavior in the form of rapid transitions between a closed and an open conductance level. The average duration of a current "burst," as well as the total time a channel is actually open within a burst, decreases with increasing Vm. Analysis of these data suggests that, over a range of Vm, increases in the rate constants for transitions from the open to the closed states largely account for the decline in macroscopic conductance with increasing Vm. Increases in rate constants for transitions from a closed to an open conductance state are more likely to account for the increase in macroscopic conductance with increasing bath pH. Since several diphtheria toxin fragments and mutants are currently available, each containing various portions of the B45 region, it may be possible to study the relationship of the structure of these complex proteins to the detailed gating properties of the ion channels that they form.

Diphtheria Toxin↗
PubMed2005

Focused electric field across the voltage sensor of potassium channels.

Voltage-gated ion channels respond to changes in membrane potential by movement of their voltage sensors across the electric field between cytoplasmic and extracellular solutions. The principal voltage sensors in these proteins are positively charged S4 segments. The absolute magnitude of S4 movement discriminates two competing classes of gating models. In one class, the movement is <10 Angstrom due to the fact that the electric field is focused by aqueous crevices in the channel protein. In an alternative model, based in part on the crystal structure of a potassium channel, the side chains of S4 arginines move their charges across the bilayer's electric field, a distance of >25 Angstrom. Here, using tethered charges attached to an S4 segment, we provide evidence that the electric field falls across a distance of <4 Angstrom, supporting a model in which the relative movement between S4 and the electric field is very small.

Animals↗
PubMed1985

Mechanisms of apparent affinity variation of guarded receptors.

Studies of interactions between blocking agents such as antiarrhythmic drugs and gated ion channels have suggested apparent variations in binding site affinity. In this report, we develop a model by which these properties of ion channels can be explained simply on the fact that blocking agent access to the channel receptor is regulated by the channel gating apparatus. We view ion channel blockade as a two stage process: diffusion of drug to a region near the channel binding site and coupling of drug to the binding site resulting in a non-conducting, drug complexed channel. We define a guarded receptor as a receptor where binding site access is regulated by some process such as gated control of the ligand diffusion path. Relationships for equilibrium properties of guarded receptors differ from those of traditional unguarded receptors. With periodic activation of channel gates, as in cardiac muscle, a true equilibrium may never be reached due to transient receptor access. Lack of equilibrium can complicate analysis. For gated ion channels we derive equilibrium block properties under conditions of no stimulation. We show that a fixed affinity guarded receptor appears as an apparent variable affinity receptor in gated channels. These results are consistent with observations of apparent variations in receptor affinity derived from studies of local anesthetics in cardiac muscle. Furthermore, by separating drug diffusion and receptor binding, this description may facilitate quantitative characterization of local anesthetics and antiarrhythmia drugs in excitable membranes.

Animals↗
PubMed1995

alpha-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability.

The hippocampal nicotinic acetylcholine receptor (nAChR) is a newly identified ligand-gated ion channel that is blocked by the snake toxin alpha-bungarotoxin (alpha-BGT) and that probably contains the alpha 7 nAChR subunit in its structure. Here its ion selectivity was characterized and compared with that of the N-methyl-D-aspartate (NMDA) receptor channel. The reversal potentials (VR) of acetylcholine- and NMDA-activated whole-cell currents were determined under various ionic conditions. Using ion activities and a Goldman-Hodgkin-Katz equation for VR shifts in the presence of Ca2+, permeability ratios were calculated. For the alpha-BGT-sensitive nAChR, PNa/PCs was close to 1 and Cl- did not contribute to the currents. Changing the [Ca2+]0 from 1 to 10 mM, the VRs of the nAChR and NMDA currents were shifted by +5.6 +/- 0.4 and +8.3 +/- 0.4 mV, respectively, and the nAChR current decay was accelerated. These shifts yielded PCa/PCss of 6.1 +/- 0.5 for the nAChR channel and 10.3 +/- 0.7 for the NMDA channel. Thus, the neuronal alpha-BGT-sensitive nAChR is a cation channel considerably selective to Ca2+ and may mediate a fast rise in intracellular Ca2+ that would increase in magnitude with membrane hyperpolarization.

Acetylcholine↗
PubMed2004

Cooperative effect of S4-S5 loops in domains D3 and D4 on fast inactivation of the Na+ channel.

Cytoplasmic S4-S5 loops have been shown to be involved in fast inactivation of voltage-gated ion channels. We studied mutations in these loops and their potential cooperative effects in domains D3 (N1151C, A1152C, I1160C/A) and D4 (F1473C, L1482C/A) of the human skeletal muscle Na(+) channel alpha-subunit (hNa(v)1.4) using expression in tsA201 cells and the whole cell patch-clamp technique. All cysteine mutations were accessible to intracellularly applied sulfhydryl reagents which considerably destabilized fast inactivation. For different combinations of corresponding D3/D4 double mutations, fast inactivation could be almost completely removed. Thermodynamic cycle analysis indicated an additive effect for N1151C/F1473C and a significant cooperative effect for I1160/L1482 double mutations. Application of oxidizing reagents such as Cu-phenanthroline to link two cysteines via a disulfide bridge did not reveal evidence for a direct physical interaction of cysteines in D3 and D4. In addition to the pronounced alterations of fast inactivation, mutations of I1160 shifted steady-state activation in the hyperpolarizing direction and slowed the kinetics of both activation and deactivation. Sulfhydryl reagents had charge-dependent effects on I1160C suggesting interaction with negative charges in another protein region. We conclude that fast inactivation of the Na(+) channel involves both S4-S5 loops in D3 and D4 in a cooperative manner. D3/S4-S5 also plays an important role in activation and deactivation.

Amino Acid Sequence↗
PubMed2004

Drosophila melanogaster GRD and LCCH3 subunits form heteromultimeric GABA-gated cation channels.

In addition to its action as a fast inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is thought to mediate excitatory action by activating cation currents in some cell types in invertebrates. However, to date no GABA receptor capable of mediating such action has been identified at the molecular level in insects. Using a systematic expression screening approach, we found that the Drosophila ligand-gated ion channel subunits GRD and LCCH3 combine to form cation-selective GABA-gated ion channels when coexpressed in Xenopus laevis oocytes. The heteromultimeric receptor is activated by GABA (EC50=4.5 microm), muscimol (EC50=4.8 microm) and trans-4-aminocrotonic acid (EC50=104.5 microm), and partially by cis-4-aminocrotonic acid (EC50=106.3 microm). Picrotoxin effectively blocked the GABA-gated channel (IC50=0.25 microm), but bicuculline, TPMTA, dieldrin and lindane did not. The benzodiazepines flunitrazepam and diazepam did not potentiate the GABA-evoked current. Our data suggest that heteromultimeric channels composed of GRD and LCCH3 subunits form GABA-gated cation channels in insects.

Animals↗
PubMed1992

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel, VDAC.

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found in Neurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 micrograms/ml) increases the voltage dependence of VDAC channels over 2-3-fold. At higher protein concentrations (50-100 micrograms/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifier in vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.

Electrophysiology↗
PubMed1993

The cyclic nucleotide-activated conductance in olfactory cilia: effects of cytoplasmic Mg2+ and Ca2+.

Olfactory receptor neurons depolarize in response to odorants. This depolarization is mediated by an increase in intracellular cyclic AMP, which directly gates channels in the membranes of the neuronal cilia. Previous evidence suggests that a Ca2+ influx during the odorant response may ultimately play a role in terminating the response. One way Ca2+ inside the cell could terminate the odorant response would be to directly inhibit the cAMP-gated channels. In this report the effects of cytoplasmic Ca2+ and Mg2+ on the cAMP-activated current were measured in single olfactory cilia. Near the neuronal resting potential, cytoplasmic Ca2+ and Mg2+ only slightly reduced the cAMP-activated current. Even at high levels (1.0 mM Ca2+ or 5.0 mM Mg2+), the average inhibition was only around 20%. It is therefore unlikely that an influx of divalent cations terminates the odorant response by a direct effect on the cAMP-gated channels.

Ambystoma↗
PubMed1993

Application of the one- and two-dimensional Ising models to studies of cooperativity between ion channels.

The Ising model of statistical physics provides a framework for studying systems of protomers in which nearest neighbors interact with each other. In this article, the Ising model is applied to the study of cooperative phenomena between ligand-gated ion channels. Expressions for the mean open channel probability, rho o, and the variance, sigma 2, are derived from the grand partition function. In the one-dimensional Ising model, interactions between neighboring open channels give rise to a sigmoidal rho o versus concentration curve and a nonquadratic relationship between sigma 2 and rho o. Positive cooperativity increases the slope at the midpoint of the rho o versus concentration curve, shifts the apparent binding affinity to lower concentrations, and increases the variance for a given rho o. Negative cooperativity has the opposite effects. Strong negative cooperativity results in a bimodal sigma 2 versus rho o curve. The slope of the rho o versus concentration curve increases linearly with the number of binding sites on a protomer, but the sigma 2 versus rho o relationship is independent of the number of ligand binding sites. Thus, the sigma 2 versus rho o curve provides unambiguous information about channel interactions. In the two-dimensional Ising model, rho o and sigma 2 are calculated numerically from a series expansion of the grand partition function appropriate for weak interactions. Virtually all of the features exhibited by the one-dimensional model are qualitatively present in the two-dimensional model. These models are also applicable to voltage-gated ion channels.

Animals↗
PubMed2006

Insect-selective spider toxins targeting voltage-gated sodium channels.

The voltage-gated sodium (Na(v)) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Na(v) channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Na(v) channel conductance. Magi 2 and Tx4(6-1) slow Na(v) channel inactivation via an interaction with site-3. The delta-palutoxins, and most likely mu-agatoxins and curtatoxins, target site-4. However, their action is complex with the mu-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion beta-toxins, but with both delta-palutoxins and mu-agatoxins slowing Na(v) channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as delta-atracotoxins, are known to target both insect and vertebrate Na(v) channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped.

Amino Acid Sequence↗
PubMed1995

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Amino Acid Sequence↗
PubMed1999

Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv. syringae, in planar lipid membranes.

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.

Amino Acid Sequence↗
PubMed2006

The reliability of relative anion-cation permeabilities deduced from reversal (dilution) potential measurements in ion channel studies.

Measurements of anion-cation permeability ratios (e.g., PCl/PNa) are most readily made by measuring changes in zero-current reversal potential when the salt concentration on one side of the membrane (e.g., external NaCl) is decreased. This is particularly useful for measuring changes in ion selectivity in wild-type and mutant channels, such as those of the ligand-gated ion channel superfamily, and has shown that many of these channels have a significant permeability to counter-ions. One Brownian dynamics study of ion permeation through such narrow ion channels failed to observe such counter-ion movement, although later, another Brownian dynamics study did observe counter-ion movement through simulations of the same channels. The question has been raised as to the reliability of such reversal potential measurements for determining permeability ratios, particularly given the use of an equation such as the Goldman-Hodgkin-Katz (GHK) equation, which is often used to calculate such ratios. A new derivation of the GHK equation in terms of activity coefficients is also included. The application of irreversible thermodynamics will be shown to qualitatively support the reliability of such experimental anion-cation permeability values derived from reversal potential measurements. It will then be shown that for such zero-current situations, different electrodiffusion models, with very different underlying assumptions, produce almost identical relative permeabilities (and reversal potentials). Finally, the results of the two Brownian dynamics simulation studies and the relationship between reversal potentials and relative permeability will be discussed.

Anions↗
PubMed1993

Brief-lifetime, fast-inactivating ion channels account for the alpha-bungarotoxin-sensitive nicotinic response in hippocampal neurons.

Single-channel currents underlying the various types of nicotinic receptor-gated whole-cell currents (previously termed IA, IB, II and III) were identified in rat hippocampal neurons. In response to applied acetylcholine (ACh), most of the neurons showed a fast-decaying whole-cell current (type IA) that can be blocked by alpha-bungarotoxin (alpha-BGT). In these neurons, a novel nicotinic receptor channel was found, having a conductance of 73 pS and an open time of 0.12 ms at -80 mV. This channel showed a fast concentration-dependent inactivation that had a time constant of 0.5 ms at 1 mM ACh. A high Ca2+ permeability and the involvement of alpha 7 receptor subunits in the channel structure were suggested.

Acetylcholine↗