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The Journal of General Physiology Mar 2023Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should...
Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should be path-independent. Here, we identify conditions to measure steady-state charge-voltage curves and show that these are curves are not hysteretic. Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left-shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms, which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea. 2017. Channels. https://doi.org/10.1080/19336950.2016.1243190). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 s) test pulses. Our study identifies a modified gating current recording protocol to obtain steady-state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.
Topics: Potassium Channels; Membrane Potentials; Ion Channel Gating; Shaker Superfamily of Potassium Channels; Oocytes
PubMed: 36692860
DOI: 10.1085/jgp.202112883 -
Proceedings of the National Academy of... Oct 2018A primary goal of sleep research is to understand the molecular basis of sleep. Although some sleep/wake-promoting circuits and secreted substances have been identified,...
A primary goal of sleep research is to understand the molecular basis of sleep. Although some sleep/wake-promoting circuits and secreted substances have been identified, the detailed molecular mechanisms underlying the regulation of sleep duration have been elusive. Here, to address these mechanisms, we developed a simple computational model of a cortical neuron with five channels and a pump, which recapitulates the cortical electrophysiological characteristics of slow-wave sleep (SWS) and wakefulness. Comprehensive bifurcation and detailed mathematical analyses predicted that leak K channels play a role in generating the electrophysiological characteristics of SWS, leading to a hypothesis that leak K channels play a role in the regulation of sleep duration. To test this hypothesis experimentally, we comprehensively generated and analyzed 14 KO mice, and found that impairment of the leak K channel () decreased sleep duration. Based on these results, we hypothesize that leak K channels regulate sleep duration in mammals.
Topics: Animals; Brain Waves; Mice; Mice, Knockout; Potassium Channels; Sleep Stages
PubMed: 30224462
DOI: 10.1073/pnas.1806486115 -
Biophysical Journal Oct 2015In the first issue, on the first page of the Biophysical Journal in 1960, Cole and Moore provided the first confirmation of the Hodgkin and Huxley formulation of the...
In the first issue, on the first page of the Biophysical Journal in 1960, Cole and Moore provided the first confirmation of the Hodgkin and Huxley formulation of the sodium and potassium conductances that underlie the action potential. In addition, working with the squid giant axon, Cole and Moore noted that strong hyperpolarization preceding a depolarizing voltage-clamp pulse delayed the rise of the potassium conductance: once started, the time course of the rise was always the same but after significant hyperpolarization there was a long lag before the rise began. This phenomenon has come to be known as the Cole-Moore effect. Their article examines and disproves the hypothesis that the lag reflects the time required to refill the membrane with potassium ions after the ions are swept out of the membrane into the axoplasm by hyperpolarization. The work by Cole and Moore indirectly supports the idea of a membrane channel for potassium conductance. However, the mechanism of the Cole-Moore effect remains a mystery even now, buried in the structure of the potassium channel, which was completely unknown at the time.
Topics: Animals; Axons; Biophysics; Decapodiformes; Drosophila; History, 20th Century; Humans; Membrane Potentials; Models, Neurological; Patch-Clamp Techniques; Potassium; Potassium Channels; Sodium
PubMed: 26445430
DOI: 10.1016/j.bpj.2015.07.052 -
International Journal of Molecular... May 2019Controlling body temperature is a matter of life or death for most animals, and in mammals the complex thermoregulatory system is comprised of thermoreceptors,... (Review)
Review
Controlling body temperature is a matter of life or death for most animals, and in mammals the complex thermoregulatory system is comprised of thermoreceptors, thermosensors, and effectors. The activity of thermoreceptors and thermoeffectors has been studied for many years, yet only recently have we begun to obtain a clear picture of the thermosensors and the molecular mechanisms involved in thermosensory reception. An important step in this direction was the discovery of the thermosensitive transient receptor potential (TRP) cationic channels, some of which are activated by increases in temperature and others by a drop in temperature, potentially converting the cells in which they are expressed into heat and cold receptors. More recently, the TWIK-related potassium (TREK) channels were seen to be strongly activated by increases in temperature. Hence, in this review we want to assess the hypothesis that both these groups of channels can collaborate, possibly along with other channels, to generate the wide range of thermal sensations that the nervous system is capable of handling.
Topics: Animals; Humans; Potassium Channels, Tandem Pore Domain; TRPV Cation Channels; Thermosensing
PubMed: 31091651
DOI: 10.3390/ijms20102371 -
Scientific Reports May 2021Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K ion diffusion across the plasma membrane, regulating both resting and action...
Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.
Topics: Action Potentials; Animals; CHO Cells; Cricetulus; Humans; Ion Channel Gating; Models, Molecular; Molecular Conformation; Patch-Clamp Techniques; Potassium Channels; Protein Binding; Protein Interaction Domains and Motifs; Spectrum Analysis; Structure-Activity Relationship
PubMed: 34021177
DOI: 10.1038/s41598-021-90002-2 -
Proceedings of the National Academy of... Jun 2020Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K channels (TREK-1) and reversibly induce loss of...
Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K channels (TREK-1) and reversibly induce loss of consciousness. For 100 y, anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of phospholipase D2 (PLD2) localization to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in whole-cell patch-clamp recordings. Localization of PLD2 renders the TRAAK channel sensitive, a channel that is otherwise anesthetic insensitive. General anesthetics, such as chloroform, isoflurane, diethyl ether, xenon, and propofol, disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLD flies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.
Topics: Anesthetics, Inhalation; Animals; Cell Membrane; Chloroform; Drosophila; Drosophila Proteins; Isoflurane; Phosphatidic Acids; Phospholipase D; Potassium Channels; Potassium Channels, Tandem Pore Domain
PubMed: 32467161
DOI: 10.1073/pnas.2004259117 -
Neuroscience Bulletin Oct 2018General anesthesia is an unconscious state induced by anesthetics for surgery. The molecular targets and cellular mechanisms of general anesthetics in the mammalian... (Review)
Review
General anesthesia is an unconscious state induced by anesthetics for surgery. The molecular targets and cellular mechanisms of general anesthetics in the mammalian nervous system have been investigated during past decades. In recent years, K channels have been identified as important targets of both volatile and intravenous anesthetics. This review covers achievements that have been made both on the regulatory effect of general anesthetics on the activity of K channels and their underlying mechanisms. Advances in research on the modulation of K channels by general anesthetics are summarized and categorized according to four large K channel families based on their amino-acid sequence homology. In addition, research achievements on the roles of K channels in general anesthesia in vivo, especially with regard to studies using mice with K channel knockout, are particularly emphasized.
Topics: Anesthetics, General; Animals; Humans; Potassium Channels
PubMed: 29948841
DOI: 10.1007/s12264-018-0239-1 -
The Journal of Neuroscience : the... Sep 2023Gain-of-function (GOF) pathogenic variants in the potassium channels KCNQ2 and KCNQ3 lead to hyperexcitability disorders such as epilepsy and autism spectrum disorders....
Gain-of-function (GOF) pathogenic variants in the potassium channels KCNQ2 and KCNQ3 lead to hyperexcitability disorders such as epilepsy and autism spectrum disorders. However, the underlying cellular mechanisms of how these variants impair forebrain function are unclear. Here, we show that the R201C variant in KCNQ2 has opposite effects on the excitability of two types of mouse pyramidal neurons of either sex, causing hyperexcitability in layer 2/3 (L2/3) pyramidal neurons and hypoexcitability in CA1 pyramidal neurons. Similarly, the homologous R231C variant in KCNQ3 leads to hyperexcitability in L2/3 pyramidal neurons and hypoexcitability in CA1 pyramidal neurons. However, the effects of KCNQ3 gain-of-function on excitability are specific to superficial CA1 pyramidal neurons. These findings reveal a new level of complexity in the function of KCNQ2 and KCNQ3 channels in the forebrain and provide a framework for understanding the effects of gain-of-function variants and potassium channels in the brain. KCNQ2/3 gain-of-function (GOF) variants lead to severe forms of neurodevelopmental disorders, but the mechanisms by which these channels affect neuronal activity are poorly understood. In this study, using a series of transgenic mice we demonstrate that the same KCNQ2/3 GOF variants can lead to either hyperexcitability or hypoexcitability in different types of pyramidal neurons [CA1 vs layer (L)2/3]. Additionally, we show that expression of the recurrent KCNQ2 GOF variant R201C in forebrain pyramidal neurons could lead to seizures and SUDEP. Our data suggest that the effects of KCNQ2/3 GOF variants depend on specific cell types and brain regions, possibly accounting for the diverse range of phenotypes observed in individuals with KCNQ2/3 GOF variants.
Topics: Animals; Mice; Gain of Function Mutation; KCNQ2 Potassium Channel; Mice, Transgenic; Neurodevelopmental Disorders; Potassium Channels; Prosencephalon; Pyramidal Cells; KCNQ3 Potassium Channel
PubMed: 37607817
DOI: 10.1523/JNEUROSCI.0980-23.2023 -
The Biochemical Journal Aug 2022Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α...
Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of β and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and β1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA-PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of β1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified β1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.
Topics: Humans; Ion Channel Gating; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channels
PubMed: 35851603
DOI: 10.1042/BCJ20210628 -
Biomolecules Sep 2020Pulmonary arterial hypertension (PAH) is a rare and severe cardiopulmonary disease without curative treatments. PAH is a multifactorial disease that involves genetic... (Review)
Review
Pulmonary arterial hypertension (PAH) is a rare and severe cardiopulmonary disease without curative treatments. PAH is a multifactorial disease that involves genetic predisposition, epigenetic factors, and environmental factors (drugs, toxins, viruses, hypoxia, and inflammation), which contribute to the initiation or development of irreversible remodeling of the pulmonary vessels. The recent identification of loss-of-function mutations in (KCNK3 or TASK-1) and (SUR1), or gain-of-function mutations in (SUR2), as well as polymorphisms in (Kv1.5), which encode two potassium (K) channels and two K channel regulatory subunits, has revived the interest of ion channels in PAH. This review focuses on KCNK3, SUR1, SUR2, and Kv1.5 channels in pulmonary vasculature and discusses their pathophysiological contribution to and therapeutic potential in PAH.
Topics: Animals; Drug Delivery Systems; Humans; Kv1.5 Potassium Channel; Nerve Tissue Proteins; Potassium Channels; Potassium Channels, Inwardly Rectifying; Potassium Channels, Tandem Pore Domain; Pulmonary Arterial Hypertension; Sulfonylurea Receptors
PubMed: 32882918
DOI: 10.3390/biom10091261