"Light" reading: targeting tryptophans in cyclic nucleotide-gated channels.
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Animals↗
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Results for “Ion Channel Gating”
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Time course and Ca(2+) dependence of sensitivity modulation in cyclic GMP-gated currents of intact cone photoreceptors.
We determined the Ca(2+) dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca(2+) levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K(1/2), the ligand concentration that half-saturates current, that, in turn, depend on Ca(2+). K(1/2) increases as Ca(2+) rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca(2+) with a single site without apparent cooperativity. (Ca)K(m), the Michaelis-Menten constant for Ca(2+) concentration is 857 +/- 68 nM for cGMP and 863 +/- 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca(2+) caused by sudden Ca(2+) sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 micro, Ca(2+) concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 +/- 0.95-fold with a mean time to peak of 1.25 +/- 0.23 s. We developed a model that assumes channel modulation is a pseudo-first-order process kinetically limited by free Ca(2+). Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s. Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.
Animals↗
Gating transitions in bacterial ion channels measured at 3 microns resolution.
Ion channels of high conductance (>200 pS) are widespread among prokaryotes and eukaryotes. Two examples, the Escherichia coli mechanosensitive ion channels Ec-MscS and Ec-MscL, pass currents of 125-300 pA. To resolve temporal details of conductance transitions, a patch-clamp setup was optimized for low-noise recordings at a time resolution of 3 microns (10-20 times faster than usual). Analyses of the high-resolution recordings confirm that Ec-MscL visits many subconductance states and show that most of the intersubstate transitions occur more slowly than the effective resolution of 3 micros. There is a clear trend toward longer transition times for the larger transitions. In Ec-MscS recordings, the majority of the observed full conductance transitions are also composite. We detected a short-lived (approximately 20 microns) Ec-MscS substate at 2/3 of full conductance; transitions between 2/3 and full conductance did not show fine structure and had a time course limited by the achieved resolution. Opening and closing transitions in MscS are symmetrical and are not preceded or followed by smaller, rapid currents ("anticipations" or "regrets"). Compared with other, lower-conductance channels, these measurements may detect unusually early states in the transitions from fully closed to fully open. Increased temporal resolution at the single-molecule level reveals that some elementary steps of structural transitions are composite and follow several alternative pathways, while others still escape resolution. High-bandwidth, low-noise single-channel measurements may provide details about state transitions in other high-conductance channels; and similar procedures may also be applied to channel- and nanopore-based single-molecule DNA measurements.
Bacterial Proteins↗
State-independent block of BK channels by an intracellular quaternary ammonium.
Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca(2+)- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43-57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between -200 and -100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in P(o), in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular.
Animals↗
Mechanisms of (local) anaesthetics on voltage-gated sodium and other ion channels.
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Anesthetics, Local↗
Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.
Hyperpolarization-activated cation currents, termed Ih, are non-uniformly distributed along dendritic arbors with current density increasing with increasing distance from the soma. The non-uniform distribution of Ih currents contributes to normalization of location-dependent variability in temporal integration of synaptic input, but the molecular basis for the graded HCN distribution remains to be investigated. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs) underlie Ih currents and consist of four members (HCN1-HCN4) of the gene family in mammals. In this investigation, we report that HCN2 forms a protein assembly with tamalin, S-SCAM and Mint2 scaffold proteins, using several different approaches including immunoprecipitation of rat brain and heterologously expressing cell extracts and glutathione S-transferase pull-down assays. The PDZ domain of tamalin interacts with HCN2 at both the PDZ-binding motif and the internal carboxy-terminal tail of HCN2, whereas binding of the PDZ domain of S-SCAM occurs at the cyclic nucleotide-binding domain (CNBD) and the CNBD-downstream sequence of the carboxy-terminal tail of HCN2. A protein assembly between HCN2 and Mint2 is formed by the interaction of the munc18-interacting domain of Mint2 with the CNBD-downstream sequence of HCN2. The results demonstrate that HCN2 forms a protein complex with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.
Animals↗
Two types of non-selective cation channel opened by muscarinic stimulation with carbachol in bovine ciliary muscle cells.
In the ciliary muscle, the tonic contraction requires a sustained influx of Ca2+ through the cell membrane. However, little has hitherto been known about the route(s) of Ca2+ influx in this tissue that lacks voltage-gated Ca2+ channels. To identify ion channels as the Ca2+ entry pathway we studied the effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. Experiments were carried out using pipettes filled with K+ -free solution containing 100 mm caesium aspartate, 5 mm BAPTA and 180 microm GTP (pH 7.0; the intracellular free Ca2+ concentration, [Ca2+]i = 70 nm). CCh evoked an inward current showing polarity reversal at a holding potential near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCCL and NSCCS) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li+, Na+, Cs+, Mg2+, Ca2+, Sr2+ and Ba2+, estimated by cation replacement procedures, were 0.9: 1.0: 1.5: 0.2: 0.3: 0.4: 0.5 for NSCCL, and 1.0: 1.0: 1.8: 2.5: 2.6: 3.2: 5.0 for NSCCS. NSCCS, but not NSCCL, was strongly inhibited by elevation of [Ca2+]i. Both NSCCL and NSCCS were dose-dependently inhibited by 1-100 microm SKF96365, La3+ and Gd3+, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the initial phasic component. NSCCL and/or NSCCS may serve as a major Ca2+ entry pathway required for sustained contraction of the bovine ciliary muscle. RT-PCR experiments in the bovine ciliary muscle (whole tissue) detected mRNAs of several transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor-operated cation channels.
Animals↗
Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus.
Hyperpolarization-activated channels (Ih or HCN channels) are widely expressed in principal neurons in the central nervous system. However, Ih in inhibitory GABAergic interneurons is less well characterized. We examined the functional properties of Ih in fast-spiking basket cells (BCs) of the dentate gyrus, using hippocampal slices from 17- to 21-day-old rats. Bath application of the Ih channel blocker ZD 7288 at a concentration of 30 microm induced a hyperpolarization of 5.7 +/- 1.5 mV, an increase in input resistance and a correlated increase in apparent membrane time constant. ZD 7288 blocked a hyperpolarization-activated current in a concentration-dependent manner (IC50, 1.4 microm). The effects of ZD 7288 were mimicked by external Cs+. The reversal potential of Ih was -27.4 mV, corresponding to a Na+ to K+ permeability ratio (PNa/PK) of 0.36. The midpoint potential of the activation curve of Ih was -83.9 mV, and the activation time constant at -120 mV was 190 ms. Single-cell expression analysis using reverse transcription followed by quantitative polymerase chain reaction revealed that BCs coexpress HCN1 and HCN2 subunit mRNA, suggesting the formation of heteromeric HCN1/2 channels. ZD 7288 increased the current threshold for evoking antidromic action potentials by extracellular stimulation, consistent with the expression of Ih in BC axons. Finally, ZD 7288 decreased the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in hippocampal granule cells, the main target cells of BCs, to 70 +/- 4% of the control value. In contrast, the amplitude of mIPSCs was unchanged, consistent with the presence of Ih in inhibitory terminals. In conclusion, our results suggest that Ih channels are expressed in the somatodendritic region, axon and presynaptic elements of fast-spiking BCs in the hippocampus.
Action Potentials↗
Queer channels in hippocampal basket cells: h-current without sag.
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Action Potentials↗
Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels.
To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.
Animals↗
Hyperpolarization-activated cyclic nucleotide-gated cation channels regulate auditory coincidence detection in nucleus laminaris of the chick.
Coincidence detection of bilateral acoustic signals in nucleus laminaris (NL) is the first step in azimuthal sound source localization in birds. Here, we demonstrate graded expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels along the tonotopic axis of NL and its role in the regulation of coincidence detection. Expression of HCN1 and HCN2, but not HCN3 or HCN4, was detected in NL. Based on measurement of both subtype mRNA and protein, HCN1 varied along the tonotopic axis and was minimal in high-characteristic frequency (CF) neurons. In contrast, HCN2 was evenly distributed. The resting conductance was larger and the steady-state activation curve of Ih was more positive in neurons of middle to low CF than those of high CF, consistent with the predominance of HCN1 channels in these neurons. Application of 8-Br-cAMP or noradrenaline generated a depolarizing shift of the Ih voltage activation curve. This shift was larger in neurons of high CF than in those of middle CF. The shift in the activation voltage of Ih depolarized the resting membrane, accelerated the EPSP time course, and significantly improved the coincidence detection in neurons of high CF, suggesting that Ih may improve the localization of sound sources.
Animals↗
Clustering of cyclic-nucleotide-gated channels in olfactory cilia.
Olfactory cilia contain the known components of olfactory signal transduction, including a high density of cyclic-nucleotide-gated (CNG) channels. CNG channels play an important role in mediating odor detection. The channels are activated by cAMP, which is formed by a G-protein-coupled transduction cascade. Frog olfactory cilia are 25-200 microm in length, so the spatial distribution of CNG channels along the length should be important in determining the sensitivity of odor detection. We have recorded from excised cilia and modeled diffusion of cAMP into a cilium to determine the spatial distribution of the CNG channels along the ciliary length. The proximal segment, which in frog is the first 20% of the cilium, appears to express a small fraction of the CNG channels, whereas the distal segment contains the majority, mostly clustered in one region.
Animals↗
Activity-dependent modulation of rod photoreceptor cyclic nucleotide-gated channels mediated by phosphorylation of a specific tyrosine residue.
Cyclic nucleotide-gated (CNG) channels are crucial for phototransduction in vertebrate rod photoreceptors. The cGMP sensitivity of these channels is modulated by diffusible intracellular messengers, including Ca2+/calmodulin, contributing to negative feedback during sensory adaptation. Membrane-associated protein tyrosine kinases and phosphatases also modulate rod CNG channels, but whether this results from direct changes in the phosphorylation state of the channel protein has been unclear. Here, we show that bovine rod CNG channel alpha-subunits (bRET) contain a tyrosine phosphorylation site crucial for modulation. bRET channels expressed in Xenopus oocytes exhibit modulation, whereas rat olfactory CNG channels (rOLF) do not. Chimeric channels reveal that differences in the C terminus, containing the cyclic nucleotide-binding domain, account for this difference. One specific tyrosine in bRET (Y498) appears to be crucial; replacement of this tyrosine in bRET curtails modulation, whereas installation into rOLF confers modulability. As the channel becomes dephosphorylated, there is an increase in the rate of spontaneous openings in the absence of ligand, indicating that changes in the phosphorylation state affect the allosteric gating equilibrium. Moreover, we find that dephosphorylation, which favors channel opening, requires open channels, whereas phosphorylation, which promotes channel closing, requires closed channels. Hence, modulation by changes in tyrosine phosphorylation is activity-dependent and may constitute a positive feedback mechanism, contrasting with negative feedback systems underlying adaptation.
Adenosine Triphosphate↗
Channelopathies: episodic disorders of the nervous system.
The field of channelopathies is a newly recognized group of disorders named after the site of their molecular defects--voltage- and ligand-gated ion channels. While voltage-gated ion channel mutants have been recognized for some time in organisms such as Drosophila, the first channelopathy in humans was reported within the last decade. The recognition of this group of disorders began with the definition of the molecular basis of a group of unusual muscle disorders called the periodic paralysis and non-dystrophic myotonias. Interestingly, this group of muscle disorders share some interesting phenotypic features with a number of seemingly disparate human diseases that involve not only skeletal muscle, but also brain and heart. Some similarities that exist among these different disorders include their episodic nature, similarities with regard to factors that precipitate attacks, therapeutic agents which can help to treat or prevent attacks, and in some cases, a degenerative component that arises in addition to the episodic attacks. The study of these diseases, along with the recognition of common clinical and pathophysiological themes among these disorders has led to tremendous growth in our understanding of these diseases and the hope of developing better therapies.
Animals↗
Prenatal ethanol exposure alters the modulation of the gamma-aminobutyric acidA1 receptor-gated chloride ion channel in adult rat offspring.
We examined the effect of prenatal ethanol exposure on gamma-aminobutyric acid (GABA)-stimulated 36Cl- flux. Sprague-Dawley rat dams were fed either a liquid diet containing 5% ethanol, pair-fed an isocalorically equivalent 0% ethanol diet or rat chow ad libitum throughout gestation. Membrane vesicles were prepared from medial frontal cortex, cerebellum and hippocampal formation of adult offspring in each diet group. GABA-stimulated 36Cl- flux was not significantly affected by prenatal ethanol exposure in any of the three brain regions examined. Positive allosteric modulation of GABA-stimulated 36Cl- flux by flunitrazepam or alphaxalone, as well as negative modulation by FG-7142 or pregnenolone, were all diminished in medial frontal cortex of 5% ethanol diet offspring compared with both ad libitum and pair-fed control groups. In cerebellum, prenatal ethanol exposure attenuated the modulatory effects of both benzodiazepines, but did not affect neurosteroid modulation. In hippocampus, prenatal ethanol exposure enhanced the effects of flunitrazepam and alphaxalone, whereas negative modulatory effects were either decreased (FG-7142) or unchanged (pregnenolone). These results indicate that moderate ethanol consumption during gestation can produce long-lasting alterations in neuromodulatory influences on GABAA receptor-mediated inhibitory neurotransmission in adult offspring. In hippocampal formation, the heightened sensitivity to positive modulatory influences may contribute to synaptic plasticity deficits in fetal ethanol-exposed rat offspring. We speculate that these prenatal ethanol-induced changes may be either a consequence of differential GABAA receptor subunit expression or receptor uncoupling in different brain regions. Furthermore, offspring exposed to ethanol in utero may display differential sensitivities to benzodiazepines and possibly other centrally active therapeutic agents.
Animals↗
Functional analysis of native and recombinant ion channels using a high-capacity nonradioactive rubidium efflux assay.
A nonradioactive cell-based rubidium (Rb(+)) efflux assay for functional analysis of native and recombinant ion channels has been developed. Cells are first loaded with rubidium, a tracer for potassium, and after channel activation, rubidium distribution between intracellular and extracellular space is determined by atomic absorption spectroscopy. The relative amount of rubidium in the cell supernatant is a direct measure of channel activity. The broad utility of the method is demonstrated by analysis of a range of different ion channels. Ligand-gated ion channels like nicotinic acetylcholine receptors and purinergic P2X receptors were studied in native PC-12 cells. Calcium-activated potassium channels were analyzed in native (small-conductance calcium-activated potassium channel, SK(Ca)) as well as recombinant cell lines (large-conductance calcium-activated potassium channel, BK(Ca)). Also recombinant voltage-gated potassium channels (Kv1.1, Kv1.4) were amenable to this functional analysis. The method is particularly useful for identification of ion channel modulators in drug discovery since it allows functional analysis with high capacity.
Animals↗
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↗
Cyclic nucleotide-gated channels: an expanding new family of ion channels.
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Animals↗