Experimental challenges in ion channel research: uncovering basic principles of permeation and gating in potassium channels

Background Ketamine shows, besides its general anesthetic effect, a local anesthetic-like action that is due to blocking of peripheral nerve sodium currents. In this study, the stereoselectivity of the blocking effects of the ketamine enantiomers S(+) and R(-) was investigated in sodium and potassium channels in peripheral nerve membranes. Methods Ion channel blockade of ketamine was investigated in enzymatically dissociated Xenopus sciatic nerves in multiple-channel and in single-channel outside-out patches. Results Concentration-effect curves for the Na+ peak current revealed half-maximal inhibiting concentrations (IC50) of 347 microM and 291 microM for S(+) and R(-) ketamine, respectively. The potential-dependent K+ current was less sensitive than the Na+ current with IC50 values of 982 microM and 942 microM. The most sensitive ion channel was the flickering background K+ channel, with IC50 values of 168 microM and 146 microM for S(+) and R(-) ketamine. Competition experiments suggest one binding site at the flicker K+ channel, with specific binding affinities for each of the enantiomers. For the Na+ channel, the block was weaker in acidic (pH = 6.6) than in neutral (pH = 7.4) and basic (pH = 8.2) solutions; for the flicker K+ channel, the block was weaker in acidic and stronger in basic solutions. Conclusions Ketamine blockade of sodium and potassium channels in peripheral nerve membranes shows no stereoselectivity except for the flicker K+ channel, which showed a very weak stereoselectivity in favor of the R(-) form. This potential-insensitive flicker K+ channel may contribute to the resting potential. Block of this channel and subsequent depolarization of the resting membrane potential leads, besides to direct Na+ channel block, to inexcitability via Na+ channel inactivation.

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Synthetic Peptide Fragments as Probes for Structure Determination of Potassium Ion-Channel Proteins

Bioscience Reports ◽

10.1023/a:1020257215577 ◽

1998 ◽

Vol 18 (6) ◽

pp. 299-312 ◽

Cited By ~ 6

Author(s):

Parvez I. Haris

Keyword(s):

Potassium Channels ◽

Ion Channel ◽

Potassium Channel ◽

Synthetic Peptide ◽

Potassium Ion ◽

Channel Structure ◽

Phospholipid Membranes ◽

Peptide Fragments ◽

2D Nmr Spectroscopy ◽

Channel Proteins

Potassium channels are a diverse class of transmembrane proteins that are responsible for diffusion of potassium ion across cell membranes. The lack of large quantities of these proteins from natural sources, is a major hindrance in their structural characterization using biophysical techniques. Synthetic peptide fragments corresponding to functionally important domains of these proteins provide an attractive approach towards characterizing the structural organization of these ion-channels. Conformational properties of peptides from three different potassium channels (Shaker, ROMK1 and minK) have been characterized in aqueous media, organic solvents and in phospholipid membranes. Techniques used for these studies include FTIR, CD and 2D-NMR spectroscopy. FTIR spectroscopy has been a particularly valuable tool for characterizing the folding of the ion-channel peptides in phospholipid membranes; the three different types of potassium channels all share a common transmembrane folding pattern that is composed of a predominantly α-helical structure. There is no evidence to suggest the presence of any significant β-sheet structure. These results are in excellent agreement with the crystal structure of a bacterial potassium channel (Doyle, D. A. et al. (1998) Science280:69–77), and suggest that all potassium channel proteins may share a common folding motif where the ion-channel structure is constructed entirely from α-helices.

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Panama: A tool for ion channel biophysics simulation

10.1101/202051 ◽

2018 ◽

Author(s):

Anuj Guruacharya

Keyword(s):

Ion Channels ◽

Potassium Channels ◽

Calcium Channels ◽

Ion Channel ◽

Chloride Channels ◽

High Threshold ◽

Spreadsheet Program ◽

Online Tool ◽

Voltage Gated ◽

Web App

I have created an online tool and an R library that simulates biophysics of voltage-gated ion channels. It is made publicly available as an R library called Panama at github.com/anuj2054/panama and as a web app at neuronsimulator.com. A need for such a tool was observed after surveying available software packages. I found that the available packages are either not robust enough to simulate multiple ion channels, too complicated, usable only as desktop software, not optimized for mobile devices, not interactive, lacking intuitive graphical controls, or not appropriate for undergraduate education. My app simulates the physiology of 11 different channels - voltage-gated sodium, potassium, and chloride channels; channels causing A-current, M-current, and After-HyperPolarization (AHP) current; calcium-activated potassium channels; low threshold T type calcium channels and high threshold L type calcium channels; leak sodium and leak potassium channels. It can simulate these channels under both current clamp and voltage clamp conditions. As we change the input values on the app, the output can be instantaneously visualized on the web browser and downloaded as a data table to be further analyzed in a spreadsheet program. The app is a first of its kind, mobile-friendly and touch-screen-friendly online tool that can be used to teach undergraduate neuroscience classes. It can also be used by researchers on their local computers as part of an R library. It has intuitive touch-optimized controls, instantaneous graphical output, and yet is pedagogically robust for education and casual research purposes.Neuroscience education, ion channel biophysics, Hodgkin-Huxley simulation, web app for neuroscience

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Speed and Noise of Neural Membranes: Ion Channel Limitations to Visual Information Transmission

Perception ◽

10.1068/v970028 ◽

1997 ◽

Vol 26 (1_suppl) ◽

pp. 38-38

Author(s):

M Weckström

Keyword(s):

Ion Channels ◽

Potassium Channels ◽

Cell Membrane ◽

Ion Channel ◽

Visual Information ◽

Electrical Response ◽

Channel Noise ◽

Maximum Speed ◽

Voltage Response ◽

Neural Noise

In dim light, photoreceptor cells and subsequent neural elements typically show high absolute sensitivity, implying that both phototransduction and synaptic transmission work at a high gain and even a single photon may produce a large electrical response. However, when there is more light, rapid adaptation at several levels of signal processing ensures that the information channel is not congested, but optimally filled with relevant voltage responses. All this is achieved by carefully tuned mechanisms that include several types of ion channels in the cell membrane. These ion-channel mechanisms have been thoroughly investigated in a few species of invertebrates and vertebrates, and some general principles are being revealed. The membrane capacitance and the resistance of the cell together define the time constant of the membrane, thus the maximum speed for building up a voltage response to light. Both in vertebrate cones and in insect microvillar photoreceptors, phototransduction takes place in an enlarged part of the cell membrane, which implies a large capacitance. This can be counteracted by making the membrane more leaky by opening more ion channels. In insect photoreceptors several types of potassium channels have been identified that perform exactly this kind of function. The types of channels vary according to the required speed of phototransduction, ie depending on the life style of the animal. In diurnal dipteran insects the potassium channels are typically of the slowly inactivating type. This channel type regulates the cell impedance according to the depolarisation caused by light stimulation. In insects active in dim environments, the potassium channels found have been predominantly rapidly inactivating. The function of this type of channels is currently under debate. In vertebrate photoreceptors several potassium channel types, including channels sensitive to intracellular calcium and pH, are expressed in the inner segments and modulate photoresponses. Opening and closing of the potassium channels also generates neural noise and thus degrades the signal-to-noise ratio (SNR). However, if the gain of phototransduction is high enough, the dominant noise comes from photon fluctuations, or from the biochemical transduction machinery, or—in some situations—from spontaneous photon-like events. Channel noise is then insignificant by comparison. Thus the optimisation of the SNR is a trade-off between bandwidth (ie speed) and amplification of the signal, and here the voltage-gated potassium channels are of prime importance.

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Neuromodulation-dependent regulation of coordinated patterns of gene expression in motor neurons of the stomatogastric ganglion

10.32469/10355/43193 ◽

2012 ◽

Author(s):

◽

Simone Temporal

Keyword(s):

Gene Expression ◽

Ion Channels ◽

Potassium Channels ◽

Calcium Channels ◽

Ion Channel ◽

Mrna Expression ◽

Stomatogastric Ganglion ◽

Muscarinic Agonist ◽

Burst Frequency ◽

Network Function

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The pyloric network of the crustacean stomatogastric ganglion (STG) is a central pattern generator that requires descending modulation for normal ongoing rhythmic activity. However, the pyloric rhythm is capable of functional recovery after removal of descending inputs. We used the STG to determine whether or not correlated mRNA ion channels are dependent on neuromodulation. Our hypothesis is that relationships between ion channels are dependent on neuromodulation, not activity. To investigate this, we first measured mRNA expression levels of three calcium channels (Ca1A, Ca1D and T-type-related channel) and two potassium channels (shal and shab), of PD cells to investigate how channel transcription may be modified to influence recovery of burst activity. We collected single PD neurons from both recovered and time-matched control preparations and quantified channel transcript levels with quantitative real-time RT-PCR. There was widespread correlation between all three calcium channels and the two potassium channels in PD cells from intact networks. Specifically, the strongest relationships were between all three calcium channels and the shal channel, which carries an A-type transient potassium current (p[less-than]0.005; R2[greater-than]0.5). Furthermore, our results show that following recovery, there are no significant changes in overall mRNA abundance across all channel types. However, there was a striking lack of any correlation between measured channel types in PD cells following recovery. These results indicate that recovered, decentralized networks do not regain rhythmicity simply by increasing or decreasing mRNA expression for a given channel or channels. In order to determine whether ion channel correlations are dependent on neuromodulation or activity, we decoupled neuromodulatory and activity inputs. We found that preparations with neuromodulatory inputs maintained relationships between mRNA channels while activity input alone did not. Further, addition of pilocarpine, the muscarinic agonist and modulator, to decentralized preparations maintained the same correlations as those found in preparations that only had neuromodulatory input. To determine whether loss of correlations affected network function, we compared the pyloric burst frequency of the different conditions. We found that the pyloric burst frequency decreased under conditions that lost correlations between ion channels due to the removal of neuromodulation. Together, these results indicate that neuromodulation maintains ion channel correlations, which are important to proper network function. They also suggest a possible novel role of neuromodulation in the regulation of gene expression.

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Protein ligands for studying ion channel proteins

The Journal of General Physiology ◽

10.1085/jgp.201711776 ◽

2017 ◽

Vol 149 (4) ◽

pp. 407-411

Author(s):

Tanmay Chavan ◽

Merritt Maduke ◽

Kenton Swartz

Keyword(s):

Potassium Channels ◽

Ion Channel ◽

Scorpion Toxin ◽

Protein Ligands ◽

Channel Proteins ◽

Ion Channel Proteins

Chavan et al. highlight work showing that a monobody can inhibit a fluoride channel using a mechanism similar to that of a scorpion toxin blocker of potassium channels.

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Ion Channel Profile of TRPM8 Cold Receptors Reveals a Role of TASK-3 Potassium Channels in Thermosensation

Cell Reports ◽

10.1016/j.celrep.2014.08.003 ◽

2014 ◽

Vol 8 (5) ◽

pp. 1571-1582 ◽

Cited By ~ 51

Author(s):

Cruz Morenilla-Palao ◽

Enoch Luis ◽

Carlos Fernández-Peña ◽

Eva Quintero ◽

Janelle L. Weaver ◽

...

Keyword(s):

Potassium Channels ◽

Ion Channel ◽

Cold Receptors

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Functional reconstitution of bacterially expressed human potassium channels in proteoliposomes: membrane potential measurements with JC-1 to assay ion channel activity

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/s0005-2736(98)00217-x ◽

1999 ◽

Vol 1416 (1-2) ◽

pp. 92-100 ◽

Cited By ~ 16

Author(s):

Baron Chanda ◽

M.K Mathew

Keyword(s):

Membrane Potential ◽

Potassium Channels ◽

Ion Channel ◽

Channel Activity

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Consensus channelome of dinoflagellates revealed by transcriptomic analysis sheds light on their physiology

ALGAE ◽

10.4490/algae.2021.36.12.2 ◽

2021 ◽

Vol 36 (4) ◽

pp. 315-326

Author(s):

Ilya Pozdnyakov ◽

Olga Matantseva ◽

Sergei Skarlato

Keyword(s):

Ion Channels ◽

Potassium Channels ◽

Ion Channel ◽

Chloride Channels ◽

Cation Channels ◽

Mechanosensitive Channels ◽

Anion Channels ◽

Voltage Gated ◽

Calcium Activated Potassium Channels ◽

Pore Domain

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (Kv), tandem Kv, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5- trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H+/Cl- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of Gprotein coupled receptors- and Ca2+-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

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Predicting the functional effects of voltage-gated potassium channel missense variants with multi-task learning

10.1101/2021.12.02.470894 ◽

2021 ◽

Author(s):

Christian Malte Boßelmann ◽

Ulrike B.S. Hedrich ◽

Peter Müller ◽

Lukas Sonnenberg ◽

Shridhar Parthasarathy ◽

...

Keyword(s):

Potassium Channels ◽

Ion Channel ◽

Neurological Diseases ◽

Predictive Performance ◽

Support Vector ◽

Loss Of Function ◽

Functional Changes ◽

Voltage Gated ◽

Task Learning ◽

Number Of Patients

AbstractPurposeVariants in genes encoding voltage-gated potassium channels are associated with a broad spectrum of neurological diseases including epilepsy, ataxia, and intellectual disability. Knowledge of the resulting functional changes, characterized as overall ion channel gain- or loss-of-function, is essential to guide clinical management including precision medicine therapies. However, for an increasing number of variants, little to no experimental data is available. New tools are needed to evaluate variant functional effects.MethodsWe catalogued a comprehensive dataset of 959 functional experiments across 19 voltage-gated potassium channels, leveraging data from 782 unique disease-associated and synthetic variants. We used these data to train a taxonomy-based multi-task learning support vector machine (MTL-SVM), and compared performance to a baseline of standard SVMs.ResultsMTL-SVM maintains channel family structure during model training, improving overall predictive performance (mean balanced accuracy 0.729 ± 0.029, AU-ROC 0.757 ± 0.039) over baseline (mean balanced accuracy 0.645 ± 0.041, AU-ROC 0.710 ± 0.074). We can obtain meaningful predictions even for channels with few known variants (KCNC1, KCNQ5).ConclusionOur model enables functional variant prediction for voltage-gated potassium channels. It may assist in tailoring current and future precision therapies for the increasing number of patients with ion channel disorders.

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