DOCSLIB.ORG

Tetrodotoxin-Insensitive Voltage-Dependent Sodium Channel (Complementary DNA/Heart Muscle/Electrophysiology/Antiarrhythmic) MARY E

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Tetrodotoxin-Insensitive Voltage-Dependent Sodium Channel (Complementary DNA/Heart Muscle/Electrophysiology/Antiarrhythmic) MARY E Proc. Natl. Acad. Sci. USA Vol. 89, pp. 554-558, January 1992 Medical Sciences Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel (complementary DNA/heart muscle/electrophysiology/antiarrhythmic) MARY E. GELLENS*, ALFRED L. GEORGE, JR.*t, LIQIONG CHENt, MOHAMED CHAHINEt, RICHARD HORNt, ROBERT L. BARCHIt§¶, AND ROLAND G. KALLENt§II Departments of *Medicine, tBiochemistry and Biophysics, ¶Neurology, and the §David Mahoney Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104; and tDepartment of Neurosciences, Roche Institute of Molecular Biology, Nutley, NJ 07110 Communicated by Eliot Stellar, September 23, 1991 ABSTRACT The principal voltage-sensitive sodium chan- MATERIALS AND METHODS nel from human heart has been cloned, sequenced, and func- tionally expressed. The cDNA, designated hH1, encodes a Screening of a Human Cardiac cDNA Library. A size- 2016-amino acid protein that is homologous to other members selected [>1 kilobase (kb)] oligo-(dT) and random-primed of the sodium channel multigene family and bears >90% adult human cardiac cDNA library constructed in AZAPII identity to the tetrodotoxin-insensitive sodium channel char- (Stratagene) was screened with cDNA probes derived from acteristic of rat heart and of immature and denervated rat rSkM2 [nucleotides (nt) 1-4385 and 5424-7076; ref. 15]. skeletal muscle. Northern blot analysis demonstrates an =9.0- Hybridizations were performed at 420C for 18 h in 50% kilobase transcript expressed in human atrial and ventricular (vol/vol) formamide/5x SSPE/5X Denhardt's solution/ cardiac muscle but not in adult skeletal muscle, brain, myo- 0.1% SDS/salmon sperm DNA (0.15 mg/ml)/random- metrium, liver, or spleen. When expressed in Xenopus oocytes, primed 32P-labeled probe (1.5 x 101 dpm/ml)] and filters were hHl exhibits rapid activation and inactivation kinetics similar washed with 6x standard saline citrate (SSC)/0.1% SDS at to native cardiac sodium channels. The single channel conduc- 650C. (lx SSPE = 0.18 M NaCl/10 mM sodium phosphate, ions is about twice that of the homol- pH 7.4/1 mM EDTA.) Plaque-purified clones were rescued tance of hHl to sodium as pBluescript phagemids and sequenced as described (15). ogous rat channel and hHl is more resistant to block by Northern Blot Analysis. Human tissues were obtained as tetrodotoxin (ICso = 5.7 pM). hHl is also resistant to Iu-cono- frozen surgical pathology specimens or procured from ca- toxin but sensitive to block by therapeutic concentrations of daveric transplant organ donors by the National Disease lidocaine in a use-dependent manner. Research Interchange (Philadelphia, PA). Total cellular RNA was isolated by the method of Chirgwin et al. (16). Samples Voltage-dependent sodium channels (NaChs) form a multi- (10 gg) were size-fractionated, electroblotted, and prehybrid- gene family, with six isoforms currently identified in the rat ized as described (15). A subtype-specific hHl antisense (1). Although structurally very similar, these isoforms can be complementary RNA probe derived from clone C92, repre- distinguished by their kinetics (2), single-channel conduc- senting 0.9 kb of the 3'-untranslated (UT) region (nt 7494- tance (3), toxin sensitivity (4, 5), and reactivity with specific 8491) was transcribed in vitro from BamHI-linearized tem- immunoreagents (6). For example, a NaCh isoform ex- plate DNA and used in Northern blot hybridizations at 5 x pressed in rat cardiac and in immature or denervated adult 106 dpm/ml. After hybridization (550C, 18 h), blots were skeletal muscle differs dramatically from the NaCh in inner- washed at a final stringency of O.lx SSC/0.1% SDS, 750C. vated skeletal muscle in its relative insensitivity to block by Functional Expression in Xenopus Oocytes. A full-length tetrodotoxin (TTX), saxitoxin, and ,u-conotoxin (4, 5, 7). hH1 construct was made in pBluescript by sequential ligation Tetrodotoxin-insensitive (TTX-I) voltage-dependent of S14 EcoRI-Sac II (nt +1 to +252), C75 Sac II-Kpn I (nt NaChs are critical for the initial rapid upstroke of the cardiac +253 to +4377), and C92 Kpn I-EcoRI (nt +4378 to +8491) action potential and are responsible for most of the Na+ fragments and the full-length hH1 insert moved into a mod- current that occurs in mammalian heart (8, 9). In addition, ified pSP64T vector (14). nt -151 to -8 of the 5'-UT region this TTX-I NaCh isoform is the principal target of class I were deleted from the construct using exonuclease III and (cardiac) antiarrhythmic agents (10). Although NaCh block- mung bean nuclease (14). A 901-base-pair (bp) Kpn I-Eag I ing agents of this type are widely used in the acute therapy of fragment from clone C21 (nt 4378-5279) was exchanged for ventricular tachyarrhythmias, their precise molecular mech- the corresponding fragment in pSP64T-hHl to eliminate a anism of action remains unclear, in part due to the difficulty cloning artifact in C92. Synthetic sense mRNA was tran- of studying human cardiac NaChs in isolation. scribed in vitro from Spe I-linearized hH1 template as de- An in vitro expression system for studying isolated human scribed (14). cardiac NaChs would have great utility. However, previous Stage V or VI oocytes were microinjected with 30-50 ng of attempts to functionally express cardiac TTX-I NaChs in synthetic hH1 mRNA and studied after 3-10 days. Na+ oocytes using RNA from mammalian heart have met with currents were measured either by two-microelectrode volt- variable success (11-13). While functional expression of the age clamp (14) or by patch clamp with outside-out patches (3). rat muscle TTX-I isoform (rSkM2) from its cloned cDNA has The bath solution contained 116 mM NaCl, 2 mM KCI, 1.8 been accomplished (14), the applicability of these findings to mM CaCl2, 2 mM MgCI2, and 5 mM Hepes (pH 7.6). For the homologous human cardiac NaCh is unknown. We report here the cloning, sequencing, and functional expression of Abbreviations: NaCh, sodium channel; TTX, tetrodotoxin; TTX-I, the TTX-I NaCh from human heart, designated hHl.** TTX-insensitive; UT, untranslated; ID, interdomain; I-V, current- voltage; nt, nucleotide(s). 'To whom reprint requests should be addressed at: 233 Anatomy The publication costs of this article were defrayed in part by page charge Chemistry, 36th & Hamilton Walk, Philadelphia, PA 19104-6059. payment. This article must therefore be hereby marked "advertisement" **The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M77235). 554 Downloaded by guest on September 27, 2021 Medical Sciences: Gellens et at. Proc. Natl. Acad. Sci. USA 89 (1992) 555 HH1I MAN F - - --LPGSFRTELAERAKAGSTESELPEARLLASKPLGPELGPELPYQTFWKKTFEFSATNALYVLS 11 5 S kM2 MAU L - - LRTSRFRSAIKMEORGASERGQEARODOSKPLGPRLGPELPYYKFVNGTFRFSATNALYVLS 1 16 SkM1 MASSSLPHLVPPGPHCLRPFTPESLAAIEQRAVEEEAR---LORNKOMEIEEPERKPRSOLEAGKNLPL IYGDPPPEVI&IPLEDLDPYYSDKKTFIVLUKGKAI F&FSATPALYLLS 115 0 H H1 PFHPVRRAAVK ILVHSL FN4LI1MCT I L TCVFMAQHOPPPWTKYVEYT FTA[IY T FEStVKILARAFCLHAFTFLRrPWNWLDF SVI IM1AYTTEFVDLGNVSALRTIFEVLRALKT ISVISG 235 56kM2 P FHPVRRAAVKI LVHSLFSM4L IMCT I L TNCVFMAA0HDPPPUWTCVVEYT FTAI OTFESLVKI LARGFCLHMAFTFLRDPWMULDFSVI VNAYTTEFVDLGNVSALRTFRVLRALKT IZSVI SG 236 SkM1 PFSIVRRVAIKVL IHALFSMF IN! TILTNCVFMTMSNPPSWSKHVEYTFTGIYTFESLIKILARiGFCIDDFTFLRDPLIMULDFSVITMAYVTEFVOLGUISALRTFRVLRALrTITVIPG 235 [Si 152- 153 1S4 0 0 0 0 H HI LKTIVGALIOSVKKLADVKVLTVFCLSVFALIGLOLFMGNLRHKCVRMFTALMGTN0GSVEAOGL--------- VWESD -----------LYLSDPEHYLLKIISTSDVLLCG 327 SkM2 LKTIVGALIOSVKKLADHMAVLTVFCLSVFALIGLQLFM4GNLRHKCVRMFTELMGTMGSVE&DGL --------- VWMLD-----------VYLNDPAHTLLKNGTTDVLLCG 328 SkM1 LKTIVGAIO KLDMLVCSFALVLL GLOCRPPNTTWGOWSDWGDTYNTNOSANTDEYNENYLGNALG 355 I55 0 KH1H NSSDAGTCPEGYHCLKAGENPDHGYTSFDSFAWAFLALFRLMTQDCWERLYOQTLRSAGK!TMI FFNLVI FLOSFYLVNLI LAVVANAYEEONDATIAETEEKEKRFQEAMEMLKKEHEA 447 SkM2 NSSDAGTCPEGYRCLKAGENPDHGYTSFDSFAWAFLALFQLMTQDCWERLYQQTLRSAJGK[YMI FFNLVIFLGSFYLVNLI LAVVANAkYEEQNQATIAETEEKEIKRFOEAMEMLKKEHEA 448 S k Ml NSSDAGHCPEGYECIKAGRNPNYGYTSYDTFSWAFLALFRLMTQDYWENLFOLTLRAAGKTYMIFFVVI IFLGSFYL1HLILAVVAMAYAEQMEATLAEDOEKEEEFQQMLEKYKKHOEE 475 SB HK1 LT IRGVDTVSRSSLENSPLAPVNSHERRSKRRKRMSSGTEECGODRLPKSDSEDGPRAMNHLSLTRGLSRTSNKPRSSRGS1 FTFRRRDLGSEADFADDENSTARESESHHTSLLVPWPL 567 S kM2 IT IRGVOTVSRSSLEN4SPLAPVTNHERKSKR-KRLSSGTEDGGDORLPKSOSEDGPRALNOLSLTHGLSRTSMRPRSSRGSI FTFRRRDOGSEADFADDENSTAGESESHRTSLLVPWPL 567 SkM1 LE--------------KAKEAAALESGEE -----------------------------------------490 H HI RR TSAOGOPSPGITSAPGHAL HGKKNSTVDCNGVVSLLGAGDPEATSPGSHLLRPVMLEHAPPOT TTPSEEPGGPQMLITSOAPC VDGFEEPGARQRALSAVSVLTSALEEL EESRHKCPPCW 687 SkM2 R HPSAQGQPGPGASAPGYVL NGKRNSTVDCP*GVVSLLGAGDAEATSPGSYLLRAPMVLDR PPDT TTPSEEPGGPOML1TPOAPCAQGFEEPGARQRALSAVSVLTSALEELEESHRKCPPCU 687 SkM ----A000PTHNK---------DCNG--SLDASGEKGPPRPS ----------------- C--------SADSAISDAMEELEEAHQKCPPWW 542 0 0 HH1 NRLAQRYLIWECCPLWMSIKQGVKLVVMOPFTDLTITNCIVLNTLFMALEHYNMTSEFEEHLOVGNLVFTGIFTAEMTFKI 1ALDPYYYFOQOWNIFDSIIVILSLKELGLSRMSNLSVL 807 5k MZ NRFAOHYLIWECCPLUHSIKQKVKFVVMDPFADLTITMCIVLNTLFM4ALEHYNMTAEFEEMLOVGNJLVFTGIFTAEMTFKI IALDPYYYFQgGWNIFDSI IVILSLMELGLSRN4GNLSVL 807 SkMl VKCAHKVLIWNCCAPWVKFKHI IYLIVMDPFVDLGITICIVLNTLFMAMEHYPMTEHFDNVLSVGHLVFTGIF1'AEMVLKLIAMDPYEYFOOGWNIFDSFIVTLSLVELGLANVQGLSVL 662 MI~ 11S2 11S3 0 H H1 RSFRLLRtVFKLAKSWPTLMTLIKIIGNSVGALGNLTLVLAI IVFIFAVVQ.MOLFGKHYSELRO--SDSGLLPRWHMMDFFHAFLIIFRILCGEWIETNWDCMEVSGQSLCLLVFLLVMVI 925 5k Z RS F RL LRVF KLAKSWPTLN4TL IK I IGNSVGALGML
Load more

Recommended publications
  • Tetrodotoxin
    Niharika Mandal et al. / Journal of Pharmacy Research 2012,5(7),3567-3570 Review Article Available online through ISSN: 0974-6943 http://jprsolutions.info Tetrodotoxin: An intriguing molecule Niharika Mandal*, Samanta Sekhar Khora, Kanagaraj Mohanapriya, and Soumya Jal School of Biosciences and Technology, VIT University, Vellore-632013 Tamil Nadu, India Received on:07-04-2012; Revised on: 12-05-2012; Accepted on:16-06-2012 ABSTRACT Tetrodotoxin (TTX) is one of the most potent neurotoxin of biological origin. It was first isolated from puffer fish and it has been discovered in various arrays of organism since then. Its origin is still unclear though some reports indicate towards microbial origin. TTX selectively blocks the sodium channel, inhibiting action potential thereby, leading to respiratory paralysis. TTX toxicity is mainly caused due to consumption of puffer fish. No Known antidote for TTX exists. Treatment is symptomatic. The present review is therefore, an effort to give an idea about the distribution, origin, structure, pharmacol- ogy, toxicity, symptoms, treatment, resistance and application of TTX. Key words: Tetrodotoxin, Neurotoxin, Puffer fish. INTRODUCTION One of the most intriguing biotoxins isolated and described in the twentieth cantly more toxic than TTX. Palytoxin and maitotoxin have potencies nearly century is the neurotoxin, Tetrodotoxin (TTX, CAS Number [4368-28-9]). 100 times that of TTX and Saxitoxin, and all four toxins are unusual in being A neurotoxin is a toxin that acts specifically on neurons usually by interact- non-proteins. Interestingly, there is also some evidence for a bacterial bio- ing with membrane proteins and ion channels mostly resulting in paralysis.
    [Show full text]
  • Suppression of Potassium Conductance by Droperidol Has
    Anesthesiology 2001; 94:280–9 © 2001 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Suppression of Potassium Conductance by Droperidol Has Influence on Excitability of Spinal Sensory Neurons Andrea Olschewski, Dr.med.,* Gunter Hempelmann, Prof., Dr.med., Dr.h.c.,† Werner Vogel, Prof., Dr.rer.nat.,‡ Boris V. Safronov, P.D., Ph.D.§ Background: During spinal and epidural anesthesia with opi- Naϩ conductance.5–8 The sensitivity of different compo- oids, droperidol is added to prevent nausea and vomiting. The nents of Naϩ current to droperidol has further been mechanisms of its action on spinal sensory neurons are not studied in spinal dorsal horn neurons9 by means of the well understood. It was previously shown that droperidol se- 10,11 lectively blocks a fast component of the Na؉ current. The au- “entire soma isolation” (ESI) method. The ESI Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/94/2/280/403011/0000542-200102000-00018.pdf by guest on 25 September 2021 thors studied the action of droperidol on voltage-gated K؉ chan- method allowed a visual identification of the sensory nels and its effect on membrane excitability in spinal dorsal neurons within the spinal cord slice and further pharma- horn neurons of the rat. cologic study of ionic channels in their isolated somata Methods: Using a combination of the patch-clamp technique and the “entire soma isolation” method, the action of droperi- under conditions in which diffusion of the drug mole- -dol on fast-inactivating A-type and delayed-rectifier K؉ chan- cules is not impeded by the connective tissue surround nels was investigated.
    [Show full text]
  • Biological Toxins Fact Sheet
    Work with FACT SHEET Biological Toxins The University of Utah Institutional Biosafety Committee (IBC) reviews registrations for work with, possession of, use of, and transfer of acute biological toxins (mammalian LD50 <100 µg/kg body weight) or toxins that fall under the Federal Select Agent Guidelines, as well as the organisms, both natural and recombinant, which produce these toxins Toxins Requiring IBC Registration Laboratory Practices Guidelines for working with biological toxins can be found The following toxins require registration with the IBC. The list in Appendix I of the Biosafety in Microbiological and is not comprehensive. Any toxin with an LD50 greater than 100 µg/kg body weight, or on the select agent list requires Biomedical Laboratories registration. Principal investigators should confirm whether or (http://www.cdc.gov/biosafety/publications/bmbl5/i not the toxins they propose to work with require IBC ndex.htm). These are summarized below. registration by contacting the OEHS Biosafety Officer at [email protected] or 801-581-6590. Routine operations with dilute toxin solutions are Abrin conducted using Biosafety Level 2 (BSL2) practices and Aflatoxin these must be detailed in the IBC protocol and will be Bacillus anthracis edema factor verified during the inspection by OEHS staff prior to IBC Bacillus anthracis lethal toxin Botulinum neurotoxins approval. BSL2 Inspection checklists can be found here Brevetoxin (http://oehs.utah.edu/research-safety/biosafety/ Cholera toxin biosafety-laboratory-audits). All personnel working with Clostridium difficile toxin biological toxins or accessing a toxin laboratory must be Clostridium perfringens toxins Conotoxins trained in the theory and practice of the toxins to be used, Dendrotoxin (DTX) with special emphasis on the nature of the hazards Diacetoxyscirpenol (DAS) associated with laboratory operations and should be Diphtheria toxin familiar with the signs and symptoms of toxin exposure.
    [Show full text]
  • Animal Venom Derived Toxins Are Novel Analgesics for Treatment Of
    Short Communication iMedPub Journals 2018 www.imedpub.com Journal of Molecular Sciences Vol.2 No.1:6 Animal Venom Derived Toxins are Novel Upadhyay RK* Analgesics for Treatment of Arthritis Department of Zoology, DDU Gorakhpur University, Gorakhpur, UP, India Abstract *Corresponding authors: Ravi Kant Upadhyay Present review article explains use of animal venom derived toxins as analgesics of the treatment of chronic pain and inflammation occurs in arthritis. It is a [email protected] progressive degenerative joint disease that put major impact on joint function and quality of life. Patients face prolonged inappropriate inflammatory responses and bone erosion. Longer persistent chronic pain is a complex and debilitating Department of Zoology, DDU Gorakhpur condition associated with a large personal, mental, physical and socioeconomic University, Gorakhpur, UttarPradesh, India. burden. However, for mitigation of inflammation and sever pain in joints synthetic analgesics are used to provide quick relief from pain but they impose many long Tel: 9838448495 term side effects. Venom toxins showed high affinity to voltage gated channels, and pain receptors. These are strong inhibitors of ion channels which enable them as potential therapeutic agents for the treatment of pain. Present article Citation: Upadhyay RK (2018) Animal Venom emphasizes development of a new class of analgesic agents in form of venom Derived Toxins are Novel Analgesics for derived toxins for the treatment of arthritis. Treatment of Arthritis. J Mol Sci. Vol.2 No.1:6 Keywords: Analgesics; Venom toxins; Ion channels; Channel inhibitors; Pain; Inflammation Received: February 04, 2018; Accepted: March 12, 2018; Published: March 19, 2018 Introduction such as the back, spine, and pelvis.
    [Show full text]
  • Chapter 25 Mechanisms of Action of Antiepileptic Drugs
    Chapter 25 Mechanisms of action of antiepileptic drugs GRAEME J. SILLS Department of Molecular and Clinical Pharmacology, University of Liverpool _________________________________________________________________________ Introduction The serendipitous discovery of the anticonvulsant properties of phenobarbital in 1912 marked the foundation of the modern pharmacotherapy of epilepsy. The subsequent 70 years saw the introduction of phenytoin, ethosuximide, carbamazepine, sodium valproate and a range of benzodiazepines. Collectively, these compounds have come to be regarded as the ‘established’ antiepileptic drugs (AEDs). A concerted period of development of drugs for epilepsy throughout the 1980s and 1990s has resulted (to date) in 16 new agents being licensed as add-on treatment for difficult-to-control adult and/or paediatric epilepsy, with some becoming available as monotherapy for newly diagnosed patients. Together, these have become known as the ‘modern’ AEDs. Throughout this period of unprecedented drug development, there have also been considerable advances in our understanding of how antiepileptic agents exert their effects at the cellular level. AEDs are neither preventive nor curative and are employed solely as a means of controlling symptoms (i.e. suppression of seizures). Recurrent seizure activity is the manifestation of an intermittent and excessive hyperexcitability of the nervous system and, while the pharmacological minutiae of currently marketed AEDs remain to be completely unravelled, these agents essentially redress the balance between neuronal excitation and inhibition. Three major classes of mechanism are recognised: modulation of voltage-gated ion channels; enhancement of gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission; and attenuation of glutamate-mediated excitatory neurotransmission. The principal pharmacological targets of currently available AEDs are highlighted in Table 1 and discussed further below.
    [Show full text]
  • Current, TTX-Resistant Na؉ Current, and Ca2؉ Current in the Action Potentials of Nociceptive Sensory Neurons
    The Journal of Neuroscience, December 1, 2002, 22(23):10277–10290 Roles of Tetrodotoxin (TTX)-Sensitive Na؉ Current, TTX-Resistant Na؉ Current, and Ca2؉ Current in the Action Potentials of Nociceptive Sensory Neurons Nathaniel T. Blair and Bruce P. Bean Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 20114 Nociceptive sensory neurons are unusual in expressing carry the largest inward charge during the upstroke of the voltage-gated inward currents carried by sodium channels re- nociceptor action potential (ϳ58%), with TTX-sensitive sodium sistant to block by tetrodotoxin (TTX) as well as currents carried channels also contributing significantly (ϳ40%), especially near by conventional TTX-sensitive sodium channels and voltage- threshold, and high voltage-activated calcium currents much dependent calcium channels. To examine how currents carried less (ϳ2%). Action potentials had a prominent shoulder during by each of these helps to shape the action potential in small- the falling phase, characteristic of nociceptive neurons. TTX- diameter dorsal root ganglion cell bodies, we voltage clamped resistant sodium channels did not inactivate completely during cells by using the action potential recorded from each cell as the action potential and carried the majority (58%) of inward the command voltage. Using intracellular solutions of physio- current flowing during the shoulder, with high voltage-activated logical ionic composition, we isolated individual components of calcium current also contributing significantly (39%).
    [Show full text]
  • Saxitoxin and ,U-Conotoxins (Brain/Electric Organ/Heart/Tetrodotoxin) EDWARD MOCZYDLOWSKI*, BALDOMERO M
    Proc. Nati. Acad. Sci. USA Vol. 83, pp. 5321-5325, July 1986 Neurobiology Discrimination of muscle and neuronal Na-channel subtypes by binding competition between [3H]saxitoxin and ,u-conotoxins (brain/electric organ/heart/tetrodotoxin) EDWARD MOCZYDLOWSKI*, BALDOMERO M. OLIVERAt, WILLIAM R. GRAYt, AND GARY R. STRICHARTZt *Department of Physiology and Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0576; tDepartment of Biology, University of Utah, Salt Lake City, UT 84112; and tAnesthesia Research Laboratories and the Department of Pharmacology, Harvard Medical School, Boston, MA 02115 Communicated by Norman Davidson, March 17, 1986 ABSTRACT The effect oftwo pL-conotoxin peptides on the 22 amino acids with amidated carboxyl termini (18). One of specific binding of [3H]saxitoxin was examined in isolated these toxins, GIIIA, has recently been shown to block muscle plasma membranes of various excitable tissues. pt-Conotoxins action potentials (18) and macroscopic Na current in a GITIA and GIHIB inhibit [3H]saxitoxin binding inlEkctrophorus voltage-clamped frog muscle fiber (19). At the single channel electric organ membranes with similar Kds of %50 x 10-9 M level, the kinetics of GIIIA block have been shown to in a manner consistent with direct competition for a common conform to a single-site binding model (Kd, 110 x 10-9 M at binding site. GITIA and GIIIB similarly compete with the 0 mV), from analysis of the statistics of discrete blocking majority (80-95%) of [3Hlsaxitoxin binding sites in rat skeletal events induced in batrachotoxin-activated Na channels from muscle with Kds of -25 and "140 x 10-9 M, respectively.
    [Show full text]
  • Evidence That Tetrodotoxin and Saxitoxin Act at a Metal Cation Binding Site in the Sodium Channels of Nerve Membrane (Solubilized Membrane/Receptors/Surface Charge) R
    Proc. Nat. Acad. Sci. USA 71 Vol. 71, No. 10, pp. 3936-3940, October 1974 Evidence That Tetrodotoxin and Saxitoxin Act at a Metal Cation Binding Site in the Sodium Channels of Nerve Membrane (solubilized membrane/receptors/surface charge) R. HENDERSON*, J. M. RITCHIE, AND G. R. STRICHARTZt Departments of Molecular Biophysics and Biochemistry, and of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Frederic M. Richards, June 17, 1974 ABSTRACT The effects of monovalent, divalent, and STX. These experiments suggest how the toxins exert their trivalent cations on the binding of tetrodotoxin and saxi- toxin to intact nerves and to a preparation of solubilized action, and provide a unifying explanation of how several nerve membranes have been examined. All eight divalent cations affect nerve membrane permeability. and trivalent cations tested, and the monovalent ions MATERIALS AND LiW, Tl+, and H+ appear to compete reversibly with the METHODS toxins for their binding site. The ability of lithium to Tritium-labeled TTX and STX were prepared and purified reduce toxin binding is paralleled by its ability to reduce (3, 5). Olfactory nerves from garfish, obtained from the Gulf tetrodotoxin-sensitive ion fluxes through the nerve mem- brane. We conclude that the toxins act at a metal cation Specimen Co., Florida, were dissected by the method of binding site in the sodium channel and suggest that this Easton (12). A detergent-solubilized extract of the nerves site is the principal coordination site for cations (normally was prepared by the method of Henderson and Wang (4). Na+ ions) as they pass through the membrane during an Binding experiments were also carried out on intact garfish action potential.
    [Show full text]
  • Alcohol Dependence and Withdrawal Impair Serotonergic Regulation Of
    Research Articles: Cellular/Molecular Alcohol dependence and withdrawal impair serotonergic regulation of GABA transmission in the rat central nucleus of the amygdala https://doi.org/10.1523/JNEUROSCI.0733-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0733-20.2020 Received: 30 March 2020 Revised: 8 July 2020 Accepted: 14 July 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Alcohol dependence and withdrawal impair serotonergic regulation of GABA 2 transmission in the rat central nucleus of the amygdala 3 Abbreviated title: Alcohol dependence impairs CeA regulation by 5-HT 4 Sophia Khom, Sarah A. Wolfe, Reesha R. Patel, Dean Kirson, David M. Hedges, Florence P. 5 Varodayan, Michal Bajo, and Marisa Roberto$ 6 The Scripps Research Institute, Department of Molecular Medicine, 10550 N. Torrey Pines 7 Road, La Jolla CA 92307 8 $To whom correspondence should be addressed: 9 Dr. Marisa Roberto 10 Department of Molecular Medicine 11 The Scripps Research Institute 12 10550 N. Torrey Pines Road, La Jolla, CA 92037 13 Tel: (858) 784-7262 Fax: (858) 784-7405 14 Email: [email protected] 15 16 Number of pages: 30 17 Number of figures: 7 18 Number of tables: 2 19 Number of words (Abstract): 250 20 Number of words (Introduction): 650 21 Number of words (Discussion): 1500 22 23 The authors declare no conflict of interest.
    [Show full text]
  • Dragon's Blood
    Available online at www.sciencedirect.com Journal of Ethnopharmacology 115 (2008) 361–380 Review Dragon’s blood: Botany, chemistry and therapeutic uses Deepika Gupta a, Bruce Bleakley b, Rajinder K. Gupta a,∗ a University School of Biotechnology, GGS Indraprastha University, K. Gate, Delhi 110006, India b Department of Biology & Microbiology, South Dakota State University, Brookings, South Dakota 57007, USA Received 25 May 2007; received in revised form 10 October 2007; accepted 11 October 2007 Available online 22 October 2007 Abstract Dragon’s blood is one of the renowned traditional medicines used in different cultures of world. It has got several therapeutic uses: haemostatic, antidiarrhetic, antiulcer, antimicrobial, antiviral, wound healing, antitumor, anti-inflammatory, antioxidant, etc. Besides these medicinal applica- tions, it is used as a coloring material, varnish and also has got applications in folk magic. These red saps and resins are derived from a number of disparate taxa. Despite its wide uses, little research has been done to know about its true source, quality control and clinical applications. In this review, we have tried to overview different sources of Dragon’s blood, its source wise chemical constituents and therapeutic uses. As well as, a little attempt has been done to review the techniques used for its quality control and safety. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Dragon’s blood; Croton; Dracaena; Daemonorops; Pterocarpus; Therapeutic uses Contents 1. Introduction ...........................................................................................................
    [Show full text]
  • Conditional Knockout of Nav1.6 in Adult Mice Ameliorates Neuropathic Pain
    www.nature.com/scientificreports OPEN Conditional knockout of NaV1.6 in adult mice ameliorates neuropathic pain Received: 22 November 2017 Lubin Chen 1,2,3, Jianying Huang1,2,3, Peng Zhao1,2,3, Anna-Karin Persson1,2,3, Accepted: 19 February 2018 Fadia B. Dib-Hajj1,2,3, Xiaoyang Cheng1,2,3, Andrew Tan1,2,3, Stephen G. Waxman1,2,3 & Published: xx xx xxxx Sulayman D. Dib-Hajj 1,2,3 Voltage-gated sodium channels NaV1.7, NaV1.8 and NaV1.9 have been the focus for pain studies because their mutations are associated with human pain disorders, but the role of NaV1.6 in pain is less understood. In this study, we selectively knocked out NaV1.6 in dorsal root ganglion (DRG) neurons, using NaV1.8-Cre directed or adeno-associated virus (AAV)-Cre mediated approaches, and examined the specifc contribution of NaV1.6 to the tetrodotoxin-sensitive (TTX-S) current in these neurons and its role in neuropathic pain. We report here that NaV1.6 contributes up to 60% of the TTX-S current in large, and 34% in small DRG neurons. We also show NaV1.6 accumulates at nodes of Ranvier within the neuroma following spared nerve injury (SNI). Although NaV1.8-Cre driven NaV1.6 knockout does not alter acute, infammatory or neuropathic pain behaviors, AAV-Cre mediated NaV1.6 knockout in adult mice partially attenuates SNI-induced mechanical allodynia. Additionally, AAV-Cre mediated NaV1.6 knockout, mostly in large DRG neurons, signifcantly attenuates excitability of these neurons after SNI and reduces NaV1.6 accumulation at nodes of Ranvier at the neuroma.
    [Show full text]
  • Siteand Mechanism of Action of Resin Acids on Voltage-Gated Ion Channels
    Linköping University Medical Dissertation No. 1620 Site and Mechanism of Action of Resin Acids on Voltage-Gated Ion Channels Malin Silverå Ejneby Department of Clinical and Experimental Medicine Linköping University, Sweden Linköping 2018 © Malin Silverå Ejneby, 2018 Cover illustration: “The Charged Pine Tree Anchored to the Ground” was designed and painted by Daniel Silverå Ejneby. Printed in Sweden by LiU-Tryck, Linköping, Sweden, 2018 ISSN: 0345-0082 ISBN: 978-91-7685-318-4 TABLE OF CONTENTS ABSTRACT ............................................................................................................................... 1 POPULÄRVETENSKAPLIG SAMMANFATTNING ................................................................. 3 LIST OF ARTICLES .................................................................................................................. 5 INTRODUCTION ....................................................................................................................... 7 Ions underlie the electrical activity in the heart and brain .................................................. 7 A mathematical model for the nerve impulse - Hodgkin and Huxley ............................... 7 Cardiac action potentials – a great diversity of shapes ...................................................... 9 Voltage-gated ion channels ......................................................................................................... 9 General structure ....................................................................................................................
    [Show full text]