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Physiology Project

Kazan State Medical University

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Barbiturates and

Chloride channel

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Barbiturates• Barbiturates are drugs that act as central nervous system depressants.• They are also effective as anxiolytics, hypnotics, and anticonvulsants.• Barbiturates vs. Benzodiazepines. • Both these highly addictive classes of drugs are medically prescribed to 

treat insomnia, anxiety, and in some cases seizures.• Derivatives of Barbituric acid or Malonylurea: Combination of urea and

malonic caid• Depressants of the central nervous system, impair or reduce activity of the brain by acting as a Gamma Amino Butyric Acid (GABA) potentiators

• Produce alcohol like symptoms such as ataxia (impaired motor control), dizziness and  slow breathing and heart rate

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Barbiturates were widely diverted from medical use and used on the street in the 60s where they were called “downers” and sold under a variety of different names.

Illicit use has declined as medical use has declined.

They had a low therapeutic index and were often used for suicide.

Marilyn Monroe died of barbiturate overdose in 1962

Barbiturates: History

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Mechanism of Action

GABA binding siteBarbiturate binding site

Barbiturates potentiate the effect of GABA at the GABA-A receptor.

The GABA-A receptor is a ligand gated ion channel membrane receptor that allows for the flow of Cl through the membrane in neurons.

GABA is the principle neurotransmitter for this receptor which upon binding causes the channel to open and creates a negative charge in the transmembrane potential.

This makes it an Inhibitory neurotransmitter

GABA

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Mechanism of ActionBarbiturates potentiate the effect of GABA by binding to the GABA-A receptor at a nearby site and increasing the chloride flow through the channel.  

      Barbiturates  also  block  the AMPA  (2-amino-3-(5-methyl-3-oxo-1,2-  oxazol-4-yl) propanoic  acid)  receptor  which  is  sensitive  to  glutamate,  the  excitatory neurotransmitter.  

      Glutamate  performs  the  opposite  effect  from  GABA  restricting  ion  flow  and increasing the transmembrane action potential of the neuron.  

        By  blocking  this  action  Barbiturates  serve  to  increase  the  duration  of  the receptor response to GABA and extend the depressed condition of the cell.

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•Treatment of INSOMNIA• Phenobarbitone  for EPILESPY • Thiopentane for ANAESTHESIA• Adjuvants in psychosomatic disorders• Pre-operative sedation• Treatment of seizure disorder

USES - BARBITURATES

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Prolongs inhibitory actions of GABA

Increases duration of ionophone

opening

Mechanism Of Action

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BARBITURATES ~ adverse effects• Residual depression after the main effect of drug has passed

• Paradoxical excitement• Hypersensitivity reaction – localized swelling of eyelid, cheek, or lip, erythematous or exfoliative dermatitis

• Synergistic action with ethanol and antihistamines

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BARBITURATES ~ TOXIC EFFECTS

• Slurred speech, ataxia, lethargy, confusion, headache, nystagmus

• CNS depression, coma, shock• Pupils first constrict and then dilate because of hypoxia• hypothermia• Cutaneous bullae• Death due to respiratory arrest of cardiovascular collapse• Chronic abuse  tolerance. 

• Withdrawal reaction: anorexia, tremor, insomnia, cramps, seizures, delirium, orthostatic hypotension

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Chloride Channel

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• Chloride channels are a functionally and structurally diverse group of anion selective channels involved in processes including the regulation of the excitability of neurones, skeletal, cardiac and smooth muscle, cell volume regulation, transepithelial salt transport, the acidification of internal and extracellular compartments, the cell cycle and apoptosis.

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Chloride channels are a superfamily of poorly understood ion channels specific for chloride. These channels may conduct many different ions, but are named for chloride because its concentration in vivo is much higher than other anions.[1] Several families of voltage-gated channels and ligand-gated channels

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A picture representation of a CLC chloride channel. The arrows indicate the orientation of each half of the individual subunit. Each CLC channel is formed from two monomers, each monomer containing the antiparallel transmembrane domain. Each monomer has its own pore through which chloride and other anions may be conducted

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A cartoon representation of a CLC channel monomer.

Two of these subunits come together to form the CLC channel. Each monomer has three binding sites for anions, Sext, Scen, and Sint. The two CBS domains bind adenosine nucleotides to alter channel function

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Cellular Cl signaling. Cells actively transport Cl across the plasma membrane by transporters that accumulate Cl intracellularly or extrude it from the cell. Cl flows passively across a variety of Cl channels in the plasma membrane, including Ca-activated Cl channels (CaCC), cAMP-activated Cl channels (CFTR), cell volume-regulated anion channels (VRAC), CLC voltage-gatedCl channels, and ligand-gated anion channels. In addition, Cl channels and transporters are found in intracellular membranes, such as the endosomal-lysosomal pathway, and play a role in regulating intravesicular pH and Cl concentration. Many proteins are regulated by Cl, as depicted by the Cl-binding protein.

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1- A, top: CLC Cl− channel model based on biochemical analysis. Conflicting results exist in the D4/D5 region. The broad hydrophobic region between D9 and D12 was difficult to investigate experimentally, but it was clear that it has an odd number of membrane crossings. The carboxy terminus of all eukaryotic CLC proteins has two CBS domains that have a so far unspecified role in protein-protein interaction. ClC-K proteins associate with the -subunit barttin, βwhich spans the membrane twice.  A,bottom: model of CLC Cl− channel derived from three-dimensional crystal structure of a bacterial CLC protein shows that the membrane-associated part of the protein is composed of 17 -helices (helix A is not inserted into the αmembrane). Inspection of the crystal reveals that most of these helices are not perpendicular to the membrane, but severely tilted.

Topology models for the established Cl− channel families

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Many of these helices do not span the width of the bilayer. This even serves an important function, as Cl− is coordinated in the pore by helices extending from either side of the membrane into the center plane. For comparison and reference, the previous nomenclature of CLC domains (D1–D12) is indicated by shaded areas and dashed lines.B: topology model of cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily. It has two blocks of six putative transmembrane spanning domains each, which are separated by a cytoplasmic region that contains the first nucleotide binding fold (NBF1) and the regulatory R domain. A second NBF is present in the carboxy terminus. It is not yet firmly established whether CFTR functions as a monomer or as a dimer.C: topology model of ligand-gated anion channels. These proteins have four transmembrane domains and assemble to homo- and heteromeric pentameric channels.

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Transepithelial transport models. A: potassium secretion in the stria vascularis of the cochlea needs basolateral Cl−channels for recycling Cl− that is transported into the cell by a Na+-K+-2Cl− cotransporter (NKCC1). K+ is secreted apically via KCNQ1/KCNE1 potassium channels. The basolateral membrane most likely contains parallel heteromeric ClC-Ka/barttin and ClC-Kb/barttin Cl− channels (147). Mutations in KCNQ1, KCNE1, NKCC1, and BSND (encoding barttin) cause deafness, but mutations in either ClC-Ka or ClC-Kb alone do not. B: chloride reabsorption in the thick ascending limb of Henle's loop involves an apical Na+-K+-2Cl− cotransporter (NKCC2) that needs a parallel K+ channel (ROMK1, Kir1.1) for recycling potassium. Cl− leaves the cell passively across the basolateral membrane through the ClC-Kb/barttin Cl−channel (147). Mutations in NKCC2, ROMK, or ClC-Kb cause variants of the same disorder, Bartter's syndrome. Mutations in the -βsubunit barttin (BSND) cause Bartter syndrome with deafness, as its loss of function affects both ClC-Ka and ClC-Kb.C: chloride secretion in intestinal crypt cells. Intracellular Cl− concentration ([Cl−]i) is raised above equilibrium by a Na+-K+-2Cl− cotransporter that needs a parallel K+ channel (KCNQ1/KCNE3) for recycling and passively leaves the cell via the apical cAMP-stimulated Cl−channel CFTR.

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Families of chloride Channels

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• The CLC family of Cl− channels in mammals. Based on homology, the nine mammalian CLC proteins can be grouped into three branches, as shown by the dendrogram (left).

• Channels of the first branch predominantly reside in the plasma membrane, whereas channels from the two other branches are thought to be predominantly intracellular.

• The localization on human chromosomes is indicated below the channel names. • The next columns indicate the most important features of their tissue distribution, their

presumed functions, the phenotype of the corresponding knock-out (KO) mouse model, and the name of the human disease associated with the channel, respectively.

• The asterisk indicates that mutations in barttin, a β-subunit for ClC-Ka and ClC-Kb , cause Bartter syndrome with sensorineural deafness and kidney failure because it compromises the function of both ClC-Ka and ClC-Kb in the kidney and the inner ear.

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The double-barreled structure of CLC channels. A: a simple model of a CLC channel. As best exemplified by theTorpedo channel ClC-0, CLC channels are believed to be dimers that have two largely independent pores. These pores can be gated individually or can be closed together by a common gate. In ClC-0, both pores have identical properties, and their individual gates are independent.  B: single-channel recordings supporting the double barrel model. Top: a recording from a native ClC-0 channel incorporated into a lipid bilayer. Note that there are long periods with zero current flow, attributed to a closed slow gate that closes both pores. An opening of this gate leads to “bursting” activity in which the equally spaced conductance levels of the individual pores become apparent. (From Miller C and Edwards EA.Chloride Channels and Carriers in Nerve, Muscle, and Glial Cells, edited by Alvárez-Leefmans FJ and Russel JM. New York: Plenum, 1990, p. 383–420.) Middle: excised patch containing a concatemer of a wild-type (WT) and a mutant (S123T) ClC-0 protein. Note that the recording can be explained by a large pore with WT conductance and a small mutant pore. In the recording to theright, bromide was substituted for chloride. As known for homomeric WT and mutant channels, WT ClC-0 conducts Cl−better than Br−, but this selectivity is lost in the mutant. This is faithfully reflected in the concatemer, showing that the permeation properties of both pores are independent. [From Ludewig et al. Bottom: registration of a ClC-0/ClC-2 concatemer. The recording can be explained by a 8.5 pS ClC-0 pore attached to a 2.5 pS ClC-∼ ∼2 pore. These values correspond to those of the corresponding homodimers, arguing even more strongly that pores are formed within the individual subunits.

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• Proximal tubular defect in endocytosis leads to secondary changes in calciotropic hormone levels and to phosphaturia in ClC-5 KO mice.

• A: mechanism leading to phosphaturia. Parathyroid hormone (PTH) is filtered into the primary urine across the glomerular filter (left). It can bind to megalin (symbolized by the zig-zag sign), which leads to its internalization and degradation in lysosomes.

• The reduced endocytosis (symbolized by hyphens) leads to an increased concentration of PTH in later parts of the proximal tubule compared with wild-type mice. This leads to an increased binding to apical PTH receptors (Y), stimulating the endocytosis of apical Na+-Pi cotransporters and their degradation in lysosomes.

• This leads to the phosphaturia observed inClcn5 − mice and in human patients with Dent's disease. B: mechanism leading to changes in vitamin D metabolism. As shown in A, the defect in endocytosis entails a luminal increase in PTH concentration, resulting in enhanced PTH signaling.

• This increases the transcription of α-hydroxylase, a mitochondrial enzyme that converts 25-hydroxyvitamin D3[25(OH)D3] to the active hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. On the other hand, the precursor 25(OH)D3, bound to its binding protein, is filtered into the primary urine and is normally endocytosed via megalin.

• This constitutes the main supply of 25(OH)D3 for the α-hydroxylase, reducing the availability of the substrate in the knockout. The supply of 25(OH)D3 is further compromised by a severe loss of this precursor into the urine that may lead to decreased serum level.

• Thus the impaired endocytosis leads to two opposing effects on the synthesis of 1,25(OH)2D3: a decrease in the precursor and an increase in enzymatic activity.

• The relative strengths of these effects determine whether there will be an increase or decrease in the serum concentration of the active hormone. An increase will lead to increased intestinal Ca2+ reabsorption and, secondarily, increased renal Ca2+ secretion, eventually causing kidney stones.

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Family of ligand-gated chloride channels. The dendrogram shows 19 members of the GABA receptor and 4 members of the glycine receptor family and their chromosomal localization. No human ortholog for the 3-subunit has been ρidentified. The chromosomal localization of the -subunit πis not known.

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• http://physrev.physiology.org/content/82/2/503.long• https://www.emory.edu/HEARTCELL/• https://en.wikipedia.org/wiki/Chloride_channel• http://physiologyonline.physiology.org/content/20/5/292• https://www.ncbi.nlm.nih.gov/pubmed/11917096

Reference

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Thank You Very Much