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PubMed2004

Epithelial sodium channel: a ligand-gated channel?

The epithelial sodium channel (ENaC) is a key component of the transepithelial Na+ transport. In epithelia, it is responsible for the maintenance of Na+ balance (which in turn controls extracellular fluid volume and arterial blood pressure) and the regulation of airway surface fluid. While the regulation of channel synthesis and surface density have been well described, the control of channel opening is still poorly understood. The channel has a large extracellular domain of as yet unknown function; a number of extracellular factors have been shown to modulate ENaC activity, including extracellular Na+ itself (through a phenomenon called 'self-inhibition'), several other organic or inorganic cations, which seem to interfere with self-inhibition, and serine proteases. Although a direct interaction with the extracellular domain of ENaC has not yet been demonstrated for each of these modulators, the available data strongly suggest that ENaC behaves as a ligand-gated channel similar to several other members of the ENaC/degenerin family.

Animals↗
PubMed1994

The conformational analysis of a synthetic S4 peptide corresponding to a voltage-gated potassium ion channel protein.

The S4 region of the Drosophila Shaker voltage-gated K+ channel has been proposed to function as a voltage-sensor. We have synthesised a peptide corresponding to this S4 region. Structural studies on the S4 peptide were conducted using Fourier transform infrared (FTIR) spectroscopy. Spectra were obtained for the peptide dissolved in aqueous solution, in trifluoroethanol solvent and also after reconstitution into lipid bilayers and micelles. The peptide in trifluoroethanol adopts an alpha-helical conformation which is in good agreement with the results of a recent 2D NMR study on the structure of a S4 peptide corresponding to the rat brain sodium channel [(1989) FEBS Lett. 257, 113-117]. A predominantly alpha-helical structure is also observed when the S4 peptide is present in aqueous lysophosphatidylcholine micelles, in dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol lipid bilayers. In contrast to this, the S4 peptide in aqueous solution is in a random coil conformation. The coil-to-helix transition observed for the S4 peptide upon its transfer from aqueous solution to lipid membrane indicates that it has a high degree of conformational flexibility and can undergo large changes in its structure in response to its environment. This may have important implications for its role in the voltage activation process during which the S4 peptide has been postulated to, at least partially, move from a lipid bilayer to an aqueous extracellular media [(1992) Biophys J. 62, 238-250]. The results of our study lend support to such a model.

Amino Acid Sequence↗
PubMed1990

Electrophysiological characterization of contact sites in brain mitochondria.

From morphological and biochemical studies it has been recognized that the regions where the outer and inner membranes of mitochondria come in close contact (contact sites) can be the route mechanism through which mitochondria interact directly with the cytoplasm. We have studied these regions electrophysiologically with the patch clamp technique, with the aim of understanding if this direct interaction is mediated by high conductance ion channels similar to the channel already detected in the inner membrane of mitochondria (Sorgato M. C., Keller, B. U., and Stühmer, W. (1987) Nature 330, 498-500). Contact sites isolated from rat brain mitochondria were thus incorporated into liposomes subsequently enlarged sufficiently to be patch clamped. This study shows that these particular fractions contain ion channels with conductances ranging from approximately 5 picosiemens to 1 nanosiemens (in symmetrical 150 mM KCl). Most of these channels are not voltage-dependent and can be open at physiological potentials sustained by respiring mitochondria. The lack of voltage sensitivity seems not to be the outcome of methodological artifacts, as voltage-gated channels are detected in giant liposomes containing either the outer mitochondrial membrane or a partially purified fraction of the inner mitochondrial membrane. These data therefore indicate that channels present in mitochondrial contact sites have properties which render them amenable to perform several of the functions hypothesized for these regions, particularly that of translocating macromolecules from the cytoplasm to the matrix of mitochondria.

Animals↗
PubMed1998

Genetic and pharmacological identification of ion channels central to the Drosophila cardiac pacemaker.

Drosophila provides an excellent model for delineating the role of ion channels in the origin and transmission of heartbeat. We report here tests in Drosophila on a wide range of mutations and pharmacological agents known to interfere with K+, Ca2+, Na+, and Cl- ion channels in well-characterized ways. We find K+ channels are central to heart function. Tetraethylammonium, which blocks all four K+ currents, slowed the heart. We were able to distinguish among these currents. The mutation slowpoke and the agent charybdotoxin, both of which affect a fast Ca(2+)-gated K+ channel, virtually eliminate heartbeat. Shaker and ether-a-go-go, which encode subunits of K+ channels, have moderate, possibly regulatory effects. "OPQ-type" Ca2+ channels are critical. omega-Conotoxin MVIIC, which blocks these channels, virtually stops the heart. Amiloride, which may affect T-type Ca2+ channels, has no effect, nor do the L-type Ca2+ blockers verapamil and diltiazem. temperature induced paralysis E, involved in the function of Na+ channels, the Na+ channel blockers tetrodotoxin and amiloride, and the Cl- blockers mefanamic and niflumic acids have no effect. Na+ and Cl- channels thus appear unnecessary for cardiac function.

Animals↗
PubMed2006

Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential.

Voltage-gated ion channels regulate many physiological functions and are targets for a number of drugs. Patch-clamp electrophysiology is the standard method for measuring channel activity because it fulfils the requirements for voltage control, repetitive stimulation and high temporal resolution, but it is laborious and costly. Here we report an electro-optical technology and automated instrument, called the electrical stimulation voltage ion probe reader (E-VIPR), that measures the activity of voltage-gated ion channels using extracellular electrical field stimulation and voltage-sensitive fluorescent probes. We demonstrate that E-VIPR can sensitively detect drug potency and mechanism of block on the neuronal human type III voltage-gated sodium channel expressed in human embryonic kidney cells. Results are compared with voltage-clamp and show that E-VIPR provides sensitive and information-rich compound blocking activity. Furthermore, we screened approximately 400 drugs and observed sodium channel-blocking activity for approximately 25% of them, including the antidepressants sertraline (Zoloft) and paroxetine (Paxil).

Cells, Cultured↗
PubMed1998

Gabapentin. Antiepileptic mechanism of action.

Gabapentin is an antiepileptic drug used in the treatment of partial and generalized tonic-clonic seizures. Its antiepileptic mechanism of action is not known. The transport of gabapentin across membranes and its demonstrated effects on voltage-gated ion channels (sodium, calcium), presynaptic mechanisms that can enhance GABAergic inhibition, and ligand-gated ion channels (GABA receptors and glutamate receptors) are reviewed.

Acetates↗
PubMed2002

A large Ca2+-dependent channel formed by recombinant ADP/ATP carrier from Neurospora crassa resembles the mitochondrial permeability transition pore.

Strong support for the central role of the ADP/ATP carrier (AAC) in the mitochondrial permeability transition (mPT) is provided by the single-channel current measurements in patch-clamp experiments with the isolated reconstituted AAC. In previous work [Brustovetsky, N., and Klingenberg, M. (1996) Biochemistry 35, 8483-8488], this technique was applied to the AAC isolated from bovine heart mitochondria. Here we used recombinant AAC (rAAC) from Neurospora crassa expressed in E. coli, since AAC from mammalian sources cannot be expresssed in E. coli. The rAAC is free from residual mitochondrial components which might associate with the AAC in preparation from bovine heart. Ca(2+)-dependent channels with up to 600 pS are obtained, which are gated at >150 mV. The channel corresponds to a preferential matrix-outside orientation of rAAC in the patch membrane as shown with carboxyatractylate and a polar gating asymmetry. The channel is inhibited by ADP and bongkrekate, not by carboxyatractylate. Cyclophilin, isolated from Neurospora crassa, suppresses the gating, thus increasing conductivity at high positive voltage. Cyclosporin A abolishes the cyclophilin effect. ADP does not eliminate the cyclophilin effect but produces fast large-amplitude flickering of the channel without a stable decrease of the channel conductance. Also the pro-oxidant tert-butyl hydroperoxide reversibly suppresses voltage gating of the channel. The results show that the AAC can be a conducting component of the mPT pore, exhibiting similar characteristics as the mPT pore (response to Ca(2+), BKA, ADP), with a cyclophilin and pro-oxidant-sensitive gating at high voltage.

Animals↗
PubMed1999

Kramers' diffusion theory applied to gating kinetics of voltage-dependent ion channels.

Kramers' diffusion theory of reaction rates in the condensed phase is considered as an alternative to the traditional discrete-state Markov (DSM) model in describing ion channel gating current kinetics. Diffusion theory can be expected to be particularly relevant in describing high-frequency (>100 kHz) events in channel activation. The generalized voltage sensor of a voltage-dependent ion channel is treated as a Brownian motion particle undergoing spatial diffusion along a one-dimensional energy landscape. Two classes of energy landscapes are considered. The first class contains large barriers, which give rise to gating currents with two distinct time scales: the usual low-frequency decay, which can modeled with a DSM scheme, and a high-frequency component arising from intrastate relaxation. Large depolarizations reduce potential barriers to such a degree that activation rates are diffusion limited, causing the two time scales to merge. Landscapes of the second class are either featureless or contain barriers that are small compared to kT; these are termed "drift landscapes." These landscapes require a larger friction coefficient to generate slow gating kinetics. The high-frequency component that appears with barrier models is not present in pure drift motion. The presence of a high-frequency component can be tested experimentally with large-bandwidth recordings of gating currents. Topics such as frequency domain analysis, spatial dependence of the friction coefficient, methods for determining the adequacy of a DSM model, and the development of physical models of gating are explored.

Diffusion↗
PubMedDate not supplied

Inherited and experimentally induced changes in gating kinetics of muscle nicotinic acetylcholine receptor.

Ligand-gated ion channels (LGIC) allow rapid responses in the nervous system. The nicotonic acetylcholine receptor (AChR) has been the model for structure-function relationship studies on this superfamily. The AChR undergoes the following functional events: 1. Binding of the neurotransmitter. 2. Opening of the ion channel. 3. Conduction of ions across the pore. 4. Desensitization. The equilibrium among these processes can be perturbed by alteration in the primary structure of the AChR or by the presence of pharmacological agents. Changes in the primary sequence leading to modifications in gating kinetics may occur in association with physiological or pathological processes. Such changes can also be genetically engineered to gain insights into structure-function relationships.

Animals↗
PubMed1995

A novel method for incorporation of ion channels into a planar phospholipid bilayer which allows solution changes on a millisecond timescale.

We have developed a method of rapidly changing the solutions on one side of a planar phospholipid bilayer. Bilayers can be painted on glass pipettes of tip diameter > or = 50 microns. By modifying an established method for rapid exchange of solutions bathing excised membrane patches, solution changes can be made at the bilayer within 10 ms. After incorporation of channels into the bilayer, the bilayer is moved into one of two parallel streams of solution flowing from a length of double-barrelled glass theta tubing. Activation of a solenoid system rapidly moves the theta tubing so that the bilayer is in the flow of the adjacent solution. For various reasons, the single-channel gating mechanisms of many channels are studied in planar bilayer systems. The conventional bilayer technique only allows for steady-state single-channel gating to be monitored. This novel method now allows the effects of rapid changes in modulators of channels incorporated into planar phospholipid bilayers to be measured.

Animals↗
PubMedDate not supplied

A cGMP-gated channel subunit in Limulus photoreceptors.

The phototransduction cascade in invertebrate photoreceptors has not been fully elucidated. It has been proposed that in Limulus ventral photoreceptor cGMP is the intracellular second messenger that directly controls the gating of the light-dependent channels (Johnson et al., 1986: Bacigalupo et al., 1991). Recently, a putative cGMP-gated channel cDNA, Lcng1, has been cloned from Limulus and shown to be expressed in the brain and the ventral eye (Chen et al., 1999). In this study, we sought to more specifically localize the LCNG1 transcript and protein. In situ hybridization was used to determine whether the gene is expressed in glia or photoreceptor cells in the ventral eye. The results clearly demonstrated that Lcngl mRNA is transcribed in the ventral photoreceptors. On Western blots probed with a polyclonal antibody raised against the C-terminus of LCNGI, a 100-kDa band and an 80-kDa band was labeled in the membrane protein preparations from brain and ventral eye, respectively. The labeling of these bands was blocked by preabsorption of the antibody with the antigen, indicating the labeling specificity. Immunocytochemistry and confocal microscopy were applied to investigate the subcellular localization of this antigen. Immunolabeling was highly localized in the transducing lobes of ventral eye photoreceptors and lateral eye photoreceptors. In both cases, the labeling was associated with membrane regions specialized for phototransduction, but the exact pattern appeared to be somewhat different in the two eyes. Preabsorption of the antiserum with antigen abolished the labeling, confirming specificity. The results lend support to the hypothesis that a cGMP-gated channel is directly involved in the phototransduction process.

Animals↗
PubMed1991

High density cAMP-gated channels at the ciliary membrane in the olfactory receptor cell.

Spatial distribution of the cAMP-gated channel was investigated in amphibian olfactory receptor cells. Low doses of cAMP applied to the cytoplasmic side of a membrane patch excised from cilia produced single channel activity of unitary conductance 28pS. Variance analysis showed that the ciliary membrane contained 920 cAMP gated-channels/microns2 in the newt and 2400 channels/microns2 in the toad. In contrast, the membrane of the dendrite and cell body contained only 2 cAMP-gated channels/microns2 (newt) and 6 channels/microns2 (toad). Thus, there is a high density of cAMP-gated channels in the cilia where olfactory transduction is thought to take place.

Animals↗
PubMed1999

Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ.

In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.

Action Potentials↗
PubMedDate not supplied

Voltage-gated potassium ion channels in colon cancer.

Voltage-gated potassium channels (VGPCs) have been previously implicated in cellular proliferation. In this study, the expression of VGPCs was examined by immunohistochemistry in seventy-four human colonic carcinoma specimens. Immunostaining for the Kv1.3 type VGPC was absent in two normal human colon specimens. Kv1.3 staining in the 74 colon cancer specimens was low in 9% (7/74), moderate in 61% (45/74) and high in 30% (22/74). Potassium channel (PC) openers, minoxidil and diazoxide (5-50 microg/ml), increased growth of SW1116, LoVo, Colo320DM and LS174t human colon cancer cell lines by 20-40%. PC-blockers, dequalinium and amiodarone, caused marked growth-inhibition of the four cell lines, at concentrations between 1 to 3 microg/ml. PC-blockers such as glibenclamide inhibited cellular proliferation at concentrations above 50 microg/ml while tetraethylammonium and 4-aminopyridine (up to 100 microg/ml) did not have significant growth-suppressive effects. Our results indicate the presence of VGPCs in colon cancer and suggest that PCs could serve as therapeutic targets.

4-Aminopyridine↗
PubMed1991

Single odor-sensitive channels in olfactory receptor neurons are also gated by cyclic nucleotides.

Olfactory transduction is thought to occur by processes that are mainly restricted to the specialized cilia emanating from the distal end of the receptor neuron's single dendrite. The involvement of a cAMP-based second messenger system seems likely, and a cyclic nucleotide-sensitive current has been recorded in patches of membrane from the cilia. However, the small diameter of the cilia and the high density of channels within the membrane limit the application of the patch recording technique in the cilia. We have found that the cAMP-sensitive channels also exist at a much lower density within the far more accessible dendritic membrane. Recording from on-cell patches, we have observed single-channel activity in response to extracellularly applied odor substances. The channels have a single-channel conductance of 40 pS and a reversal potential near 0 mV. These same channels are activated by treatments that elevate intracellular cyclic nucleotide concentrations. The results provide a direct demonstration that the cyclic nucleotide-gated channel is the conductance pathway for the odor-elicited current.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Structure and action of the nicotinic acetylcholine receptor explored by electron microscopy.

The nicotinic acetylcholine (ACh) receptor is the transmitter-gated ion channel at the nerve/muscle synapse. Electron microscopical experiments on isolated postsynaptic membranes have determined the structure of this channel and how the structure changes upon activation. When ACh enters the ligand-binding domain it initiates rotations of the protein chains on opposite sides of the entrance to the membrane-spanning pore. These rotations are communicated to the pore-lining alpha-helices and open the gate--a constricting hydrophobic girdle at the middle of the membrane--by breaking it apart. The movements are small and involve energetically favourable displacements parallel to the membrane plane.

Animals↗
PubMed2002

Differential blockade of neuronal voltage-gated Na(+) and K(+) channels by antidepressant drugs.

The effects of a range of antidepressants were investigated on neuronal voltage-gated Na(+) and K(+) channels. With the exception of phenelzine, all antidepressants inhibited batrachotoxin-stimulated 22Na(+) uptake, most likely via negative allosteric inhibition of batrachotoxin binding to neurotoxin receptor site-2 on the Na(+) channel. Imipramine also produced a differential action on macroscopic Na(+) and K(+) channel currents in acutely dissociated rat dorsal root ganglion neurons. Imipramine produced a use-dependent block of Na(+) channels. In addition, there was a hyperpolarizing shift in the voltage-dependence of steady-state Na(+) channel inactivation and slowed repriming kinetics consistent with imipramine having a higher affinity for the inactivated state of the Na(+) channel. At higher concentrations, imipramine also blocked delayed-rectifier and transient outward K(+) currents in the absence of alterations to the voltage-dependence of activation or the kinetics of inactivation. These actions on voltage-gated ion channels may underlie the therapeutic and toxic effects of these drugs.

Algorithms↗