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European Journal of Medicinal Chemistry Nov 2023Voltage-gated potassium channel K1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca...
Voltage-gated potassium channel K1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca homeostasis. Here, we present the structure-activity relationship, K1.3 inhibition, and immunosuppressive effects of new thiophene-based K1.3 inhibitors with nanomolar potency on K current in T-lymphocytes and K1.3 inhibition on Ltk cells. The new K1.3 inhibitor trans-18 inhibited K1.3 -mediated current in phytohemagglutinin (PHA)-activated T-lymphocytes with an IC value of 26.1 nM and in mammalian Ltk cells with an IC value of 230 nM. The K1.3 inhibitor trans-18 also had nanomolar potency against K1.3 in Xenopus laevis oocytes (IC = 136 nM). The novel thiophene-based K1.3 inhibitors impaired intracellular Ca signaling as well as T-cell activation, proliferation, and colony formation.
Topics: Animals; Mammals; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Structure-Activity Relationship; T-Lymphocytes; Thiophenes; Immunosuppressive Agents
PubMed: 37454520
DOI: 10.1016/j.ejmech.2023.115561 -
The Journal of Biological Chemistry Aug 2020Two-pore-domain potassium channels (K) are the major determinants of the background potassium conductance. They play a crucial role in setting the resting membrane...
Two-pore-domain potassium channels (K) are the major determinants of the background potassium conductance. They play a crucial role in setting the resting membrane potential and regulating cellular excitability. These channels form homodimers; however, a few examples of heterodimerization have also been reported. The K channel subunits TRESK and TREK-2 provide the predominant background potassium current in the primary sensory neurons of the dorsal root and trigeminal ganglia. A recent study has shown that a TRESK mutation causes migraine because it leads to the formation of a dominant negative truncated TRESK fragment. Surprisingly, this fragment can also interact with TREK-2. In this study, we determined the biophysical and pharmacological properties of the TRESK/TREK-2 heterodimer using a covalently linked TRESK/TREK-2 construct to ensure the assembly of the different subunits. The tandem channel has an intermediate single-channel conductance compared with the TRESK and TREK-2 homodimers. Similar conductance values were recorded when TRESK and TREK-2 were coexpressed, demonstrating that the two subunits can spontaneously form functional heterodimers. The TRESK component confers calcineurin-dependent regulation to the heterodimer and gives rise to a pharmacological profile similar to the TRESK homodimer, whereas the presence of the TREK-2 subunit renders the channel sensitive to the selective TREK-2 activator T2A3. In trigeminal primary sensory neurons, we detected single-channel activity with biophysical and pharmacological properties similar to the TRESK/TREK-2 tandem, indicating that WT TRESK and TREK-2 subunits coassemble to form functional heterodimeric channels also in native cells.
Topics: Animals; HEK293 Cells; Humans; Ion Transport; Mice; Neurons; Potassium; Potassium Channels; Potassium Channels, Tandem Pore Domain; Protein Multimerization; Somatosensory Cortex; Xenopus laevis
PubMed: 32641496
DOI: 10.1074/jbc.RA120.014125 -
ELife Jun 2023Voltage-gated potassium (K) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. K channel...
Voltage-gated potassium (K) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. K channel mutations lead to disordered cellular excitability. Loss-of-function mutations, for example, result in membrane hyperexcitability, a characteristic of epilepsy and cardiac arrhythmias. Interventions intended to restore K channel function have strong therapeutic potential in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise a class of K channel activators with potential applications in the treatment of arrhythmogenic disorders such as long QT syndrome (LQTS). LQTS is caused by a loss-of-function of the cardiac I channel - a tetrameric potassium channel complex formed by K7.1 and associated KCNE1 protein subunits. We have discovered a set of aromatic PUFA analogues that produce robust activation of the cardiac I channel, and a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We determine the mechanisms through which tyrosine PUFA analogues exert strong activating effects on the I channel by generating modified aromatic head groups designed to probe cation-pi interactions, hydrogen bonding, and ionic interactions. We found that tyrosine PUFA analogues do not activate the I channel through cation-pi interactions, but instead do so through a combination of hydrogen bonding and ionic interactions.
Topics: Humans; Potassium Channels; Potassium Channels, Voltage-Gated; KCNQ1 Potassium Channel; Fatty Acids, Unsaturated; Long QT Syndrome; Arrhythmias, Cardiac; Tyrosine
PubMed: 37350568
DOI: 10.7554/eLife.85773 -
Gene Sep 2015The KCNE single-span transmembrane subunits are encoded by five-member gene families in the human and mouse genomes. Primarily recognized for co-assembling with and... (Review)
Review
The KCNE single-span transmembrane subunits are encoded by five-member gene families in the human and mouse genomes. Primarily recognized for co-assembling with and functionally regulating the voltage-gated potassium channels, the broad influence of KCNE subunits in mammalian physiology belies their small size. KCNE2 has been widely studied since we first discovered one of its roles in the heart and its association with inherited and acquired human Long QT syndrome. Since then, physiological analyses together with human and mouse genetics studies have uncovered a startling array of functions for KCNE2, in the heart, stomach, thyroid and choroid plexus. The other side of this coin is the variety of interconnected disease manifestations caused by KCNE2 disruption, involving both excitable cells such as cardiomyocytes, and non-excitable, polarized epithelia. Kcne2 deletion in mice has been particularly instrumental in illustrating the potential ramifications within a monogenic arrhythmia syndrome, with removal of one piece revealing the unexpected complexity of the puzzle. Here, we review current knowledge of the function and pathobiology of KCNE2.
Topics: Animals; Arrhythmias, Cardiac; Electrophysiology; Heart; Humans; Potassium Channels, Voltage-Gated
PubMed: 26123744
DOI: 10.1016/j.gene.2015.06.061 -
Biochimica Et Biophysica Acta Oct 2015Potassium channels are a diverse group of pore-forming transmembrane proteins that selectively facilitate potassium flow through an electrochemical gradient. They... (Review)
Review
Potassium channels are a diverse group of pore-forming transmembrane proteins that selectively facilitate potassium flow through an electrochemical gradient. They participate in the control of the membrane potential and cell excitability in addition to different cell functions such as cell volume regulation, proliferation, cell migration, angiogenesis as well as apoptosis. Because these physiological processes are essential for the correct cell function, K+ channels have been associated with a growing number of diseases including cancer. In fact, different K+ channel families such as the voltage-gated K+ channels, the ether à-go-go K+ channels, the two pore domain K+ channels and the Ca2+-activated K+ channels have been associated to tumor biology. Potassium channels have a role in neoplastic cell-cycle progression and their expression has been found abnormal in many types of tumors and cancer cells. In addition, the expression and activity of specific K+ channels have shown a significant correlation with the tumor malignancy grade. The aim of this overview is to summarize published data on K+ channels that exhibit oncogenic properties and have been linked to a more malignant cancer phenotype. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.
Topics: Apoptosis; Cell Movement; Cell Proliferation; Cell Size; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Membrane Potentials; Neoplasms; Neovascularization, Pathologic; Phenotype; Potassium; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Potassium Channels, Tandem Pore Domain; Potassium Channels, Voltage-Gated
PubMed: 25517985
DOI: 10.1016/j.bbamem.2014.12.008 -
Nature Communications Jan 2023The K channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K binding sites (S1-S4). Only two sites (S3, S4) are present...
The K channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K-selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K channels. Our results establish the generality of K-induced SF conformational stability across the K channel superfamily, and introduce an approach to study manipulation of channel selectivity.
Topics: Potassium Channels; Potassium; Binding Sites; Protein Conformation
PubMed: 36609575
DOI: 10.1038/s41467-022-35756-7 -
The Journal of Physiology Jun 2015The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two... (Review)
Review
The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel.
Topics: Animals; Potassium Channels, Voltage-Gated; Protein Structure, Tertiary
PubMed: 25603957
DOI: 10.1113/jphysiol.2014.287672 -
Frontiers in Cellular and Infection... 2019develops in environments where nutrient availability, osmolarity, ionic concentrations, and pH undergo significant changes. The ability to adapt and respond to such...
develops in environments where nutrient availability, osmolarity, ionic concentrations, and pH undergo significant changes. The ability to adapt and respond to such conditions determines the survival and successful transmission of . Ion channels play fundamental roles in controlling physiological parameters that ensure cell homeostasis by rapidly triggering compensatory mechanisms. Combining molecular, cellular and electrophysiological approaches we have identified and characterized the expression and function of a novel calcium-activated potassium channel (TcCAKC). This channel resides in the plasma membrane of all 3 life stages of and shares structural features with other potassium channels. We expressed TcCAKC in oocytes and established its biophysical properties by two-electrode voltage clamp. Oocytes expressing TcCAKC showed a significant increase in inward currents after addition of calcium ionophore ionomycin or thapsigargin. These responses were abolished by EGTA suggesting that TcCAKC activation is dependent of extracellular calcium. This activation causes an increase in current and a negative shift in reversal potential that is blocked by barium. As predicted, a single point mutation in the selectivity filter (Y313A) completely abolished the activity of the channels, confirming its potassium selective nature. We have generated knockout parasites deleting one or both alleles of TcCAKC. These parasite strains showed impaired growth, decreased production of trypomastigotes and slower intracellular replication, pointing to an important role of TcCAKC in regulating infectivity. To understand the cellular mechanisms underlying these phenotypic defects, we used fluorescent probes to evaluate intracellular membrane potential, pH, and intracellular calcium. Epimastigotes lacking the channel had significantly lower cytosolic calcium, hyperpolarization, changes in intracellular pH, and increased rate of proton extrusion. These results are in agreement with previous reports indicating that, in trypanosomatids, membrane potential and intracellular pH maintenance are linked. Our work shows TcCAKC is a novel potassium channel that contributes to homeostatic regulation of important physiological processes in and provides new avenues to explore the potential of ion channels as targets for drug development against protozoan parasites.
Topics: Calcium; Cell Membrane; Chagas Disease; Cloning, Molecular; Cytoplasm; Cytosol; Electrophysiological Phenomena; Electrophysiology; Gene Expression Regulation; Gene Knockout Techniques; Hydrogen-Ion Concentration; Membrane Potentials; Mutagenesis, Site-Directed; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Sequence Analysis; Trypanosoma cruzi
PubMed: 32010643
DOI: 10.3389/fcimb.2019.00464 -
Channels (Austin, Tex.) Dec 2024K channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit... (Review)
Review
K channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit complex comprising four central pore-forming Kir6 inward rectifier potassium channel subunits surrounded by four regulatory subunits known as the sulfonylurea receptor, SUR, which confer homeostatic metabolic control of K gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for K expression and function. For more than four decades, understanding the structure-function relationship of Kir6 and SUR has remained a central objective of clinical significance. Here, we review progress in correlating the wealth of functional data in the literature with recent K cryoEM structures.
Topics: Sulfonylurea Receptors; Potassium Channels, Inwardly Rectifying; Membrane Potentials; Adenosine Triphosphate; KATP Channels
PubMed: 38489043
DOI: 10.1080/19336950.2024.2327708 -
The Journal of Biological Chemistry Dec 2017Voltage-activated human ether-á-go-go-related gene (hERG) potassium channels are critical for the repolarization of cardiac action potentials and tune-spike frequency...
Voltage-activated human ether-á-go-go-related gene (hERG) potassium channels are critical for the repolarization of cardiac action potentials and tune-spike frequency adaptation in neurons. Two isoforms of mammalian ERG1 channel subunits, ERG1a and ERG1b, are the principal subunits that conduct the I current in the heart and are also broadly expressed in the nervous system. However, there is little direct evidence that ERG1a and ERG1b form heteromeric channels. Here, using electrophysiology, biochemistry, and fluorescence approaches, we systematically tested for direct interactions between hERG1a and hERG1b subunits. We report 1) that hERG1a dominant-negative subunits suppress hERG1b currents (and vice versa), 2) that disulfide bonds form between single cysteine residues experimentally introduced into an extracellular loop of hERG1a and hERG1b subunits and produce hERG1a-hERG1b dimers, and 3) that hERG1a and hERG1b subunits tagged with fluorescent proteins that are FRET pairs exhibit robust energy transfer at the plasma membrane. Thus, multiple lines of evidence indicated a physical interaction between hERG1a and hERG1b, consistent with them forming heteromeric channels. Moreover, co-expression of variable ratios of and RNA yielded channels with deactivation kinetics that reached a plateau and were different from those of hERG1b channels, consistent with a preference of hERG1b subunits for hERG1a subunits. Cross-linking studies revealed that an equal input of hERG1a and hERG1b yields more hERG1a-hERG1a or hERG1a-hERG1b dimers than hERG1b-hERG1b dimers, also suggesting that hERG1b preferentially interacts with hERG1a. We conclude that hERG1b preferentially forms heteromeric ion channels with hERG1a at the plasma membrane.
Topics: ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Fluorescence Resonance Energy Transfer; Heart; Humans; Ion Channel Gating; Long QT Syndrome; Myocardium; Potassium Channels, Voltage-Gated; Protein Isoforms; Protein Subunits
PubMed: 29089383
DOI: 10.1074/jbc.M117.816488