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1.
Nature ; 531(7596): 656-60, 2016 Mar 31.
Article in English | MEDLINE | ID: mdl-27007841

ABSTRACT

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium. This Ca(2+)-responsive mechanosensor hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers. Here we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. We developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca(2+) influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signalling.


Subject(s)
Calcium/metabolism , Cilia/metabolism , Mechanotransduction, Cellular , Animals , Calcium/analysis , Calcium Signaling , Embryo, Mammalian/cytology , Epithelial Cells/cytology , Female , Hair Cells, Auditory, Inner/cytology , Kidney/cytology , Male , Mice , Mice, Transgenic , Models, Biological
2.
Br J Pharmacol ; 170(8): 1449-58, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24528237

ABSTRACT

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.


Subject(s)
Databases, Pharmaceutical , Molecular Targeted Therapy , Pharmacology , Humans , Ligands , Pharmaceutical Preparations/chemistry
3.
Nature ; 479(7371): 67-73, 2011 Nov 02.
Article in English | MEDLINE | ID: mdl-22051675

ABSTRACT

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCß4, a vertebrate homologue of the Drosophila NorpA phospholipase C which mediates rhabdomeric phototransduction. The Plcb4(-/-) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCß4-mediated pathway that nonetheless diverges in the two locations.


Subject(s)
Iris/metabolism , Iris/radiation effects , Light Signal Transduction/radiation effects , Mammals/physiology , Retina/metabolism , Retina/radiation effects , Rod Opsins/metabolism , Animals , Iris/anatomy & histology , Iris/cytology , Light Signal Transduction/physiology , Mice , Phospholipase C beta/metabolism , Photic Stimulation , Primates/physiology , Reflex, Pupillary/physiology , Reflex, Pupillary/radiation effects , Retina/cytology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/radiation effects
5.
J Physiol ; 586(10): 2477-86, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18356202

ABSTRACT

Two years ago, genes coding for voltage-gated proton channels in humans, mice and Ciona intestinalis were discovered. Transfection of cDNA encoding the human HVCN1 (H(V)1) or mouse (mVSOP) ortholog of HVCN1 into mammalian cells results in currents that are extremely similar to native proton currents, with a subtle, but functionally important, difference. Expressed proton channels exhibit high H(+) selectivity, voltage-dependent gating, strong temperature sensitivity, inhibition by Zn(2+), and gating kinetics similar to native proton currents. Like native channels, expressed proton channels are regulated by pH, with the proton conductance-voltage (g(H)-V) relationship shifting toward more negative voltages when pH(o) is increased or pH(i) is decreased. However, in every (unstimulated) cell studied to date, endogenous proton channels open only positive to the Nernst potential for protons, E(H). Consequently, only outward H(+) currents exist in the steady state. In contrast, when the human or mouse proton channel genes are expressed in HEK-293 or COS-7 cells, sustained inward H(+) currents can be elicited, especially with an inward proton gradient (pH(o) < pH(i)). Inward current is the result of a negative shift in the absolute voltage dependence of gating. The voltage dependence at any given pH(o) and pH(i) is shifted by about -30 mV compared with native H(+) channels. Expressed H(V)1 voltage dependence was insensitive to interventions that promote phosphorylation or dephosphorylation of native phagocyte proton channels, suggesting distinct regulation of expressed channels. Finally, we present additional evidence that speaks against a number of possible mechanisms for the anomalous voltage dependence of expressed H(+) channels.


Subject(s)
Gene Expression Regulation/physiology , Ion Channels/biosynthesis , Protons , Animals , Cell Line , Humans , Ion Channel Gating/physiology , Ion Channels/genetics , Membrane Potentials/physiology , Mice
6.
Science ; 294(5550): 2372-5, 2001 Dec 14.
Article in English | MEDLINE | ID: mdl-11743207

ABSTRACT

The pore-forming subunits of canonical voltage-gated sodium and calcium channels are encoded by four repeated domains of six-transmembrane (6TM) segments. We expressed and characterized a bacterial ion channel (NaChBac) from Bacillus halodurans that is encoded by one 6TM segment. The sequence, especially in the pore region, is similar to that of voltage-gated calcium channels. The expressed channel was activated by voltage and was blocked by calcium channel blockers. However, the channel was selective for sodium. The identification of NaChBac as a functionally expressed bacterial voltage-sensitive ion-selective channel provides insight into both voltage-dependent activation and divalent cation selectivity.


Subject(s)
Bacillus/chemistry , Bacterial Proteins , Sodium Channels/genetics , Sodium Channels/metabolism , Sodium/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Bacillus/genetics , Bacillus/metabolism , CHO Cells , COS Cells , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/chemistry , Calcium Channels/metabolism , Cricetinae , Dihydropyridines/pharmacology , Genes, Bacterial , Ion Channel Gating , Membrane Potentials , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Patch-Clamp Techniques , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Sodium Channels/chemistry , Tetrodotoxin/pharmacology , Transfection
7.
Proc Natl Acad Sci U S A ; 98(22): 12527-31, 2001 Oct 23.
Article in English | MEDLINE | ID: mdl-11675491

ABSTRACT

Calcium ions play a primary role in the regulation of sperm cell behavior. We report finding a voltage-gated ion channel (CatSper2) that is expressed in male germ cells but not in other cells. The putative channel contains 6 transmembrane segments, making it more similar to the voltage-gated potassium channels, but the ion selectivity pore domain sequence resembles that of a Ca(v) channel. The mRNA is expressed during the meiotic or postmeiotic stages of spermatogenesis, and the protein is localized to the sperm flagellum, suggesting a role in the regulation of sperm motility. The mRNA for the channel is present in mouse, rat, and human sperm cells, and the gene is found on chromosome 2 E5-F1 in the mouse and 15q13 in the human. Recently, another voltage-gated channel (CatSper) that has features similar to the one reported here was discovered. It also is expressed within the flagellum and is required for normal fertility of mice. However, expression of CatSper2 alone or coexpression with CatSper in cultured cells, or attempts to coimmunoprecipitate the two proteins from germ cells failed to demonstrate that these two unique but similar alpha-like subunits form either a homo- or heterotetramer. It is possible, therefore, that two independent alpha subunits, different from other known voltage-gated channels, regulate sperm motility.


Subject(s)
Ion Channel Gating , Ion Channels/analysis , Spermatozoa/metabolism , Amino Acid Sequence , Animals , Chromosome Mapping , Ion Channels/chemistry , Ion Channels/genetics , Male , Mice , Molecular Sequence Data , Precipitin Tests , Protein Sorting Signals/genetics , RNA, Messenger/analysis
8.
Nature ; 413(6856): 603-9, 2001 Oct 11.
Article in English | MEDLINE | ID: mdl-11595941

ABSTRACT

Calcium and cyclic nucleotides have crucial roles in mammalian fertilization, but the molecules comprising the Ca2+-permeation pathway in sperm motility are poorly understood. Here we describe a putative sperm cation channel, CatSper, whose amino-acid sequence most closely resembles a single, six-transmembrane-spanning repeat of the voltage-dependent Ca2+-channel four-repeat structure. CatSper is located specifically in the principal piece of the sperm tail. Targeted disruption of the gene results in male sterility in otherwise normal mice. Sperm motility is decreased markedly in CatSper-/- mice, and CatSper-/- sperm are unable to fertilize intact eggs. In addition, the cyclic-AMP-induced Ca2+ influx is abolished in the sperm of mutant mice. CatSper is thus vital to cAMP-mediated Ca2+ influx in sperm, sperm motility and fertilization. CatSper represents an excellent target for non-hormonal contraceptives for both men and women.


Subject(s)
Calcium Channels/physiology , Fertility/physiology , Sperm Motility/physiology , Spermatozoa/physiology , Amino Acid Sequence , Animals , Calcium/physiology , Calcium Channels/chemistry , Calcium Channels/genetics , Cloning, Molecular , Cyclic AMP/physiology , Electrophysiology , Female , Fertility/genetics , Fertilization , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Sperm Tail/metabolism , Spermatozoa/chemistry , Stem Cells
9.
Nat Rev Neurosci ; 2(6): 387-96, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11389472

ABSTRACT

Mammalian homologues of the Drosophila transient receptor potential (TRP) channel gene encode a family of at least 20 ion channel proteins. They are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. A common theme that links the TRP channels is their activation or modulation by phosphatidylinositol signal transduction pathways. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. Three subgroups comprise the TRP channel family; the best understood of these mediates responses to painful stimuli. Other proposed functions include repletion of intracellular calcium stores, receptor-mediated excitation and modulation of the cell cycle.


Subject(s)
Calcium Channels/metabolism , Calcium Channels/physiology , Calcium Signaling/physiology , Cell Membrane/metabolism , Cell Membrane/physiology , Animals , Humans , Signal Transduction/physiology , TRPC Cation Channels
10.
J Am Coll Cardiol ; 37(8): 2136-43, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11419900

ABSTRACT

OBJECTIVES: We sought to study the role of I(KACh) in atrial fibrillation (AF) and the potential electrophysiologic effects of a specific I(KACh) antagonist. BACKGROUND: I(KACh) mediates much of the cardiac responses to vagal stimulation. Vagal stimulation predisposes to AF, but the specific role of I(KACh) in the generation of AF and the electrophysiologic effects of specific I(KACh) blockade have not been studied. METHODS: Adult wild-type (WT) and I(KACh)-deficient knockout (KO) mice were studied in the absence and presence of the muscarinic receptor agonist carbachol. The electrophysiologic features of KO mice were compared with those of WT mice to assess the potential effects of a specific I(KACh) antagonist. RESULTS: Atrial fibrillation lasting for a mean of 5.7+/-11 min was initiated in 10 of 14 WT mice in the presence of carbachol, but not in the absence of carbachol. Atrial arrhythmia could not be induced in KO mice. Ventricular tachyarrhythmia could not be induced in either type of mouse. Sinus node recovery times after carbachol and sinus cycle lengths were shorter and ventricular effective refractory periods were greater in KO mice than in WT mice. There was no significant difference between KO and WT mice in AV node function. CONCLUSIONS: Activation of I(KACh) predisposed to AF and lack of I(KACh) prevented AF. It is likely that I(KACh) plays a crucial role in the generation of AF in mice. Specific I(KACh) blockers might be useful for the treatment of AF without significant adverse effects on the atrioventricular node or the ventricles.


Subject(s)
Atrial Fibrillation/physiopathology , Ion Channel Gating/physiology , Potassium Channels, Inwardly Rectifying , Potassium Channels/physiology , Animals , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Disease Models, Animal , Electrocardiography , Electrophysiologic Techniques, Cardiac , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Mice , Mice, Knockout
11.
J Immunol ; 166(10): 6126-33, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11342632

ABSTRACT

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling/immunology , Dendritic Cells/physiology , Adenosine Triphosphate/physiology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels/physiology , Calcium Signaling/drug effects , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dihydropyridines/pharmacology , Immunophenotyping , Ion Channel Gating/drug effects , Ion Channel Gating/immunology , Mice , Mice, Inbred C57BL , Patch-Clamp Techniques , Thapsigargin/pharmacology
12.
Neuron ; 29(3): 645-55, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11301024

ABSTRACT

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.


Subject(s)
Brain Chemistry , Calcium Channels/chemistry , Cation Transport Proteins , Ion Channels/chemistry , Animals , Axons/chemistry , Calcium/analysis , Calcium Channels/analysis , Calcium Channels/genetics , Cations , Cell Line , Dendrites/chemistry , Electric Conductivity , Embryo, Mammalian , Gene Expression , Hippocampus/chemistry , Humans , Kidney , Macromolecular Substances , Neurons/chemistry , Neurons/ultrastructure , Rats , Receptors, N-Methyl-D-Aspartate/physiology , TRPC Cation Channels , Transfection
13.
Nature ; 410(6829): 705-9, 2001 Apr 05.
Article in English | MEDLINE | ID: mdl-11287959

ABSTRACT

The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Animals , CHO Cells , Calcium Channels/genetics , Cricetinae , Cricetulus , Electrophysiology , Rats , TRPV Cation Channels , Transfection , Tumor Cells, Cultured
14.
Science ; 291(5506): 1043-7, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11161216

ABSTRACT

We cloned and characterized a protein kinase and ion channel, TRP-PLIK. As part of the long transient receptor potential channel subfamily implicated in control of cell division, it is a protein that is both an ion channel and a protein kinase. TRP-PLIK phosphorylated itself, displayed a wide tissue distribution, and, when expressed in CHO-K1 cells, constituted a nonselective, calcium-permeant, 105-picosiemen, steeply outwardly rectifying conductance. The zinc finger containing alpha-kinase domain was functional. Inactivation of the kinase activity by site-directed mutagenesis and the channel's dependence on intracellular adenosine triphosphate (ATP) demonstrated that the channel's kinase activity is essential for channel function.


Subject(s)
Ion Channels/genetics , Ion Channels/metabolism , Membrane Proteins , Protein Kinases/genetics , Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , CHO Cells , Calcium/metabolism , Catalytic Domain , Cations/metabolism , Cell Line , Cricetinae , DNA, Complementary , Electric Conductivity , Humans , Ion Channels/chemistry , Mice , Molecular Sequence Data , Mutation , Myelin Basic Protein/metabolism , Patch-Clamp Techniques , Phosphorylation , Protein Kinases/chemistry , Protein Serine-Threonine Kinases , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , TRPM Cation Channels , Transfection , Two-Hybrid System Techniques , Type C Phospholipases/metabolism
15.
J Biol Chem ; 276(14): 11409-13, 2001 Apr 06.
Article in English | MEDLINE | ID: mdl-11148218

ABSTRACT

G-protein-coupled inwardly rectifying K(+) (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability by hyperpolarizing the membrane potential in cardiac cells, neurons, and secretory cells. Although direct regulation of GIRKs by the heterotrimeric G-protein subunit Gbetagamma has been extensively studied, little is known about the number of Gbetagamma binding sites per channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified Gbetagamma subunits. The observed laddering pattern of Gbetagamma attachment to GIRK4 homotetramers was consistent with the binding of one, two, three, or four Gbetagamma molecules per channel tetramer. The fraction of channels chemically cross-linked to four Gbetagamma molecules increased with increasing Gbetagamma concentrations and approached saturation. These results suggest that GIRK tetrameric channels have four Gbetagamma binding sites. Thus, GIRK (Kir 3.x) channels, like the distantly related cyclic nucleotide-gated channels, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Animals , CHO Cells , COS Cells , Cattle , Cell Membrane/chemistry , Cell Membrane/metabolism , Cricetinae , Cross-Linking Reagents , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Heterotrimeric GTP-Binding Proteins/chemistry , Potassium Channels/chemistry , Protein Binding
16.
J Biol Chem ; 275(46): 36211-6, 2000 Nov 17.
Article in English | MEDLINE | ID: mdl-10956667

ABSTRACT

G-protein-gated inwardly rectifying K(+) (GIRK) channels are widely expressed in the brain and are activated by at least eight different neurotransmitters. As K(+) channels, they drive the transmembrane potential toward E(K) when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits (GIRK1-4 or Kir 3.1-4), with GIRK1 being the most unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However, more recent data indicate that native channels can be either homo- or heterotetrameric complexes composed of several GIRK subunit combinations. The functional implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK channels formed by the co-assembly of GIRK2 and GIRK3, the most abundant GIRK subunits found in the mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at the single channel level was an approximately 5-fold lower sensitivity to activation by Gbetagamma. Complexes containing only GIRK2 and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels composed of GIRK2 and GIRK3 subunits exist in brain.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels/chemistry , Potassium Channels/metabolism , Animals , Brain/metabolism , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Ion Channel Gating , Macromolecular Substances , Mice , Mice, Knockout , Patch-Clamp Techniques , Potassium Channels/genetics , Precipitin Tests , Protein Binding , Protein Structure, Quaternary , Protein Subunits , Transfection
17.
J Biol Chem ; 275(38): 29709-16, 2000 Sep 22.
Article in English | MEDLINE | ID: mdl-10900209

ABSTRACT

G protein-gated inwardly rectifying potassium (GIRK) channels are a family of K(+)-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein G beta gamma subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by G beta gamma. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables G beta gamma gating of the channel complex.


Subject(s)
GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , Heterotrimeric GTP-Binding Proteins/physiology , Ion Channel Gating/physiology , Potassium Channels/physiology , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Molecular Sequence Data , Phosphorylation , Rats
18.
J Neurosci ; 20(15): 5608-15, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10908597

ABSTRACT

Neuronal G-protein-gated potassium (K(G)) channels are activated by several neurotransmitters and constitute an important mode of synaptic inhibition in the mammalian nervous system. K(G) channels are composed of combinations of four subunits termed G protein-gated inwardly rectifying K(+) channels (GIRK). All four GIRK subunits are expressed in the brain, and there is a general consensus concerning the expression patterns of GIRK1, GIRK2, and GIRK3. The localization pattern of GIRK4, however, remains controversial. In this study, we exploit the negative background of mice lacking a functional GIRK4 gene to identify neuronal populations that contain GIRK4 mRNA. GIRK4 mRNA was detected in only a few regions of the mouse brain, including the deep cortical pyramidal neurons, the endopiriform nucleus and claustrum of the insular cortex, the globus pallidus, the ventromedial hypothalamic nucleus, parafascicular and paraventricular thalamic nuclei, and a few brainstem nuclei (e.g., the inferior olive and vestibular nuclei). Mice lacking GIRK4 were viable and appeared normal and did not display gross deficiencies in locomotor activity, visual tasks, and pain perception. Furthermore, GIRK4-deficient mice performed similarly to wild-type controls in the passive avoidance paradigm, a test of aversive learning. GIRK4 knock-out mice did, however, exhibit impaired performance in the Morris water maze, a test of spatial learning and memory.


Subject(s)
Behavior, Animal/physiology , Brain Chemistry/physiology , Maze Learning/physiology , Potassium Channels, Inwardly Rectifying , Potassium Channels/analysis , Potassium Channels/genetics , Animals , Avoidance Learning/physiology , Chick Embryo , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Gene Expression/physiology , In Situ Hybridization , Ion Channel Gating/physiology , Locomotion/physiology , Male , Memory/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/analysis
19.
Biophys J ; 79(1): 202-14, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10866948

ABSTRACT

The outer nuclear membrane, endoplasmic reticulum, and mitochondrial membrane ion channels are poorly understood, although they are important in the control of compartmental calcium levels, cell division, and apoptosis. Few direct recordings of these ion channels have been made because of the difficulty of accessing these intracellular membranes. Using patch-clamp techniques on isolated nuclei, we measured distinct ion channel classes on the outer nuclear envelope of T-cell (human Jurkat) and BFL5 cell (murine promyelocyte) lines. We first imaged the nuclear envelopes of both Jurkat and FL5 cells with atomic force microscopy to determine the density of pore proteins. The nuclear pore complex was intact at roughly similar densities in both cell types. In patch-clamp recordings of Jurkat nuclear membranes, Cl channels (105 +/- 5 pS) predominated and inactivated with negative pipette potentials. Nucleotides transiently inhibited the anion channel. In contrast, FL5 nuclear channels were cation selective (52 +/- 2 pS), were inactivated with positive membrane potentials, and were insensitive to GTPgammaS applied to the bath. We hypothesize that T- and B-cell nuclear membrane channels are distinct, and that this is perhaps related to their unique roles in the immune system.


Subject(s)
B-Lymphocytes/metabolism , Ion Channels/biosynthesis , Nuclear Envelope/metabolism , T-Lymphocytes/metabolism , Animals , Anions/metabolism , B-Lymphocytes/cytology , Cations/metabolism , Cell Line , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Chloride Channels/biosynthesis , Humans , Ion Channel Gating/physiology , Jurkat Cells , Mice , Microscopy, Atomic Force , Nuclear Envelope/ultrastructure , Nuclear Proteins/biosynthesis , Patch-Clamp Techniques , Second Messenger Systems/physiology , T-Lymphocytes/cytology
20.
J Biol Chem ; 275(17): 12363-6, 2000 Apr 28.
Article in English | MEDLINE | ID: mdl-10777517

ABSTRACT

pICln is a 26-kDa protein that is ubiquitously expressed and highly conserved from Xenopus laevis to Homo sapiens. The physiological functions of pICln remain to be established. To address this question, we disrupted the ICln gene in embryonic stem cells. We found that murine embryos lacking ICln die early in gestation (between stages E3.5 and E7.5). Furthermore, we found that ICln is essential for embryonic stem cell viability. Previously, we showed that pICln interacts directly with a homolog of a yeast protein that binds a PAK-like kinase and participates in the regulation of cell morphology and cell cycling. pICln also forms a complex with several core spliceosomal proteins, and this interaction may play a role in the regulation of spliceosomal biogenesis. Collectively, these data strongly suggest that pICln participates in critical cellular pathways, including regulation of the cell cycle and RNA processing.


Subject(s)
Cell Survival , Chloride Channels/physiology , Embryo, Mammalian/physiology , Ion Channels , 3T3 Cells , Animals , Cell Line , Chloride Channels/genetics , Gene Targeting , Genotype , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Recombination, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/physiology , Time Factors
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