Biochemistry of Neurotransmission: A Type of Cell-Cell Signaling

1
Biochemistry of Neurotransmission A Type of
Cell-Cell Signaling

• Biochemistry is fun

2
Biochemistry of Cell Signaling
Fig. 19-1
3
Study Guide

• Contrast resting, ligand-gated, voltage-gated,
  and signal-gated ion channels
• How do voltage gated ion channels monitor the
  voltage?
• What is the neurotransmitter at the vertebrate
  neuromuscular junction? The crayfish
  neuromuscular junction?
• What is the chief excitatory neurotransmitter in
  the mammalian brain? The chief inhibitory
  neurotransmitter? What vitamin is required for
  the synthesis of the chief inhibitory brain
  neurotransmitter? What is the role of PyP in
  catecholamine synthesis? What is the role of
  tetrahydrobiopterin in second messenger synthesis
  (3 answers)?
• How is the action of acetylcholine terminated?
  Serotonin?
• What is Parkinson's disease, and what is the
  mechanism for its development? How is
  parkinsonism treated?
• How are neurotransmitters released at the
  synapse? What proteins are involved? Name the
  calcium ion sensor
• Describe the Otto Loewi experiment and explain
  its significance.
• What is myasthenia gravis and what is the
  mechanism for its development?
• Describe the molecular components and actions of
  G-proteins. How many transmembrane domains do
  receptors that interact with G-proteins possess?
• What is the role of cyclic GMP in vision?

4
Overview

• The human brain contains about 1012 neurons, and
  some neurons make 1000 connections
• Dendrites, cell body, axon
• The cell body contains the nucleus, and this is
  where almost all protein synthesis occurs
• The cell body also contains nearly all of the
  lysosomes
• Proteins and other molecules are transported from
  the nucleus via axoplasmic transport
• Axons are long processes specialized for the
  conduction of action potentials
• The nervous system also contains glial cells that
  support and nourish the neurons (Schwann cells in
  the peripheral nervous system)
• Types of neurons sensory neurons, interneurons,
  motor neurons

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Neuroanatomy
Fig. 19-2
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Anatomy of the Neuron

• Arrows indicate the direction of conduction of
  the action potential
• A motor neuron typically has a single axon
• The axon of the sensory neuron branches after it
  leaves the cell body
• Both branches are structurally and functionally
  axons
• The cell body is located in the dorsal root
  ganglion near the spinal cord

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Signaling within the Neuron

• The axon carries an electrical impulse called the
  action potential.
• These move at speeds of 100 m/s
• The action potential originates in the axon
  hillock
• An axon can be 1 meter and longer (from spinal
  cord to the big toe)
• Dendrites receive signals and convert them into
  small electric impulses and transmit them to the
  cell body

8
The Action Potential

• AP transient depolarization of the membrane
  followed by repolarization to about 60 mV
• Below 1 action potential every 4 msec
• Invasion of the synapse results in release of
  neurotransmitter that bind to postsynaptic
  receptors and activate them
• This can be excitatory (depolarization)
• This can be inhibitory (hyperpolarization)

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Synapses

• Specialized Sites where neurons communicate with
  other cells
• Neurons
• Muscle cells
• Endocrine cells
• Types of synapses
• Chemical (vast, vast majority)
• Presynaptic cell contains vesicles
• The neurotransmitter (NT) interacts with
  postsynaptic cell within 0.5 ms
• Electrical (a curiosity)
• Connected by gap junctions
• The next slide illustrates various synapses
• Hippocampal interneurons which makes about 1000
  synapses (orange red dots)
• Electron micrograph of a CNS synapse

10
Synapses
11
The Action Potential and the Conduction of
Electric Impulses

• An electric potential exists across the plasma
  membrane because of ion gradients
• Resting potential is about 60 mV owing to the
  large number of open potassium channels
• Voltage-gated channels allow the transmission of
  the electrical impulses
• Action Potential
• Na channels open allowing Na to enter the cell
  and depolarize it, then they close for a
  refractory period
• K channels open permitting efflux of K which
  hyperpolarizes the membrane
• As these channels close, the membrane returns to
  its resting potential

12
Ion Channels

• (c, d) are located on dendrites and cell bodies
• d is coupled to a NT receptor via a G-protein

13
Origin of the Resting Potential

• Sodium pump or sodium/potassium ATPase generates
  these gradients
• Na is extracellular
• K is intracellular
• A- represents protein
• The open potassium channels and the potassium
  gradient are responsible for the resting potential

14
Myelination Increases the Velocity of Impulse
Conduction

• Myelin is a specialized membrane
• Derived from Schwann cells in the PNS
• Derived from oligodendrocytes (glia) in CNS
• Contains protein and lipid
• Action potential jumps from node to node
  (saltatory conduction), and this greatly
  increases the velocity of AP conduction
• Less energy is required to transmit an action
  potential in a myelinated nerve
• More energy is required to transmit an action
  potential in unmyelinated nerves
• Most nerves are myelinated

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Myelin Sheath

• (a) Myelinated peripheral nerve surrounded by a
  Schwann cell that produces the myelin
• (b) Sciatic nerve axon is surrounded by a myelin
  sheath (MS)

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Myelinated and Non-Myelinated Nerves in Dental
Pulp
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Structure of a Peripheral Myelinated Axon
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Saltatory Conduction from Node to Node

• Saltatory refers to the jumping of the action
  potential from node to node
• The nodes are the only regions along the axon
  where the axonal membrane is in direct contact
  with the extracellular fluid

19
Molecular Properties of Voltage-Gated Ion Channels

• Voltage-gated K channels are assembled from four
  similar subunits, each of which has six
  membrane-spanning alpha helices and a nonhelical
  P segment that lines the ion pore 24 TM segments
  total
• Voltage-gated Na and Ca2 channels are monomeric
  proteins containing four homologous domains each
  similar to a K channel subunit 24 TM segments
  total
• The S4 alpha helix acts as a voltage sensor
• Voltage-sensing alpha helices have a lysine or
  arginine every third or fourth residue outward
  movement toward the negative extracellular space
  in response to depolarization opens the channel
• Voltage-gated K, Na, and Ca2 channel proteins
  contain cytosolic domains that move into the open
  channel thereby inactivating it
• Non-voltage gated K channels and
  nucleotide-gated channels lack a voltage-sensing
  alpha helix, but otherwise their structures are
  very similar to the voltage-gated K channels

20
Transmembrane Structures of Gated Ion-Channel
Proteins

• The voltage-gated K channel consists of four
  identical subunits and six transmembrane alpha
  helices
• Helix 4 is the voltage sensor
• cAMP and cGMP-gated ion channels are made of four
  identical subunits that lack a voltage sensor
• These occur in the olfactory and visual systems,
  respectively

21
Voltage-gated Na Channel

• All voltage-gated channels contain four
  transmembrane domains (each with 6 TM segments),
  and each domain contributes to the central pore
• In the resting state, the gate obstructs the
  channel
• There are four voltage-sensing alpha helices
  which have positively charged side chains every
  third residue
• When the outside of the membrane becomes negative
  (depolarized) the helices move toward the outer
  plasma membrane surface causing a conformational
  change in the gate segment that opens the channel
  as shown in b
• Shortly afterwards, the helices return to the
  resting position as shown in c
• The channel inactivating segment (purple) moves
  into the open channel preventing further ion
  movement as shown in c

22
Structure and Function of the Voltage-gated Na
Channel
23
Transmembrane Structures of Gated Ion-Channel
Proteins

• Voltage-gated Na and Ca channels are monomers
• These form a channel similar to that of the K
  channel
• There are 24 transmembrane segments
• These channels contain regulatory portions, not
  shown here

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Neurotransmitters (NTs)

• Impulses are transmitted by the release of NTs
  from the axon terminal of the presynaptic cell
  into the synaptic cleft. NTs bind to specific
  receptors on the postsynaptic cell causing a
  change in the ion permeability and the potential
  of the postsynaptic plasma membrane
• Classical NTs are imported from the cytosol into
  synaptic vesicles by a protein-coupled
  antiporter, a V-type ATPase that maintains a low
  intravesicular pH (V vesicle)
• The V-type ATPase pumps protons into the synaptic
  vesicle
• Then protons leave the vesicle in exchange for
  the NT which is transported inward this is
  antiport
• Catecholamines (DA, NE, EPI) are unstable at pH
  7 they are stable at pH 5 in the intravesicular
  space
• Excitatory receptors lead to depolarization
  thereby promoting generation of an action
  potential
• Inhibitory receptors lead to hyperpolarization
  thereby inhibiting generation of an action
  potential
• Ligand-gated receptors induce rapid (msec)
  responses

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Neurotransmitters (cont)

• G-protein coupled receptors (GPCR) induce
  responses that last for seconds or more
• Removal of transmitters is by hydrolysis
  (metabolism), diffusion away from the synapse, or
  most commonly by uptake
• ACh by hydrolysis
• Nearly all other NTs by uptake
• A single postsynaptic cell can amplify, modify,
  and compute excitatory and inhibitory signals
  received from multiple presynaptic neurons
• Postsynaptic cells generate action potentials in
  an all-or-nothing fashion
• At electric synapses, ions pass directly from the
  pre to the postsynaptic cell through gap
  junctions
• Impulse transmission at chemical synapses occurs
  with a small time delay but is nearly
  instantaneous at electric synapses

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Small Molecule Neurotransmitters

• Acetylcholine (ACh)
• Vertebrate neuromuscular junction
• Pre and postganglionic parasympathetic nervous
  system
• Preganglionic sympathetic nervous system
• Central nervous system (CNS)
• Glycine chief inhibitory NT in the spinal cord
• Glutamate chief excitatory NT in the CNS
• Dopamine (DA) selected CNS neurons parkinsonism
• Norepinephrine (NE)
• Postganglionic sympathetic NS
• Selected CNS neurons

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Small Molecule Neurotransmitters (cont)

• Epinephrine
• Selected CNS
• Adrenal medulla
• 5-Hydroxytryptophan (5-HT), or serotonin CNS
  (Prozac, Zoloft, SSRIs, selective serotonin
  reuptake inhibitors)
• Histamine (mast cells)
• GABA (gamma aminobutyric acid) chief inhibitory
  NT in the CNS

28
Selected Neurotransmitters
ACh at the vertebrate nm junction Glutamate at
the invertebrate nm junction (crayfish and
lobster)
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Acetylcholine

• Grandfather of all neurotransmitters
• Sites of action
• Vertebrate neuromuscular junction nicotinic
• Pre-and post-ganglionic parasympathetic
  nicotinic and muscarinic, respectively
• Pre-ganglionic parasympathetic nicotinic
• Present in CNS (both Muscarinic and Nicotinic
  receptors)
• Inactivated by hydrolysis (the only classical
  neurotransmitter that is inactivated by
  metabolism)
• Pathology
• Alzheimer (?)

30
Acetylcholine Metabolism (Fig. 19-15, 19-16)

• ACh is inactivated by hydrolysis

31
Acetylcholine Congeners (Fig. 19-17)
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Catecholamines
33
Catecholamine Biosynthesis

• Tyrosine hydroxylase
• First and rate-limiting
• Activated by PKA and other PKs
• Uses tetrahydrobiopterin as cofactor
• Aromatic Amino Acid Decarboxylase (AAD) uses PyP
  (B6) as cofactor
• Dopamine beta hydroxylase (DBH) uses vitamin C,
  or ascorbate

34
Parkinsonism

• A slowly progressive neurological disease
  characterized by
• a fixed inexpressive face
• a tremor at rest, slowing of voluntary movements
• a gait with short accelerating steps, peculiar
  posture, and muscle weakness
• It is caused by degeneration of the basal
  ganglia, and by low production of the
  neurotransmitter dopamine
• Most patients are over 50, but at least 10
  percent are under 40
• Also known as paralysis agitans and shaking palsy
• Treatment is by medication, such as levodopa and
  carbidopa (Sinemet)
• Levodopa is converted to dopamine levodopa is
  able to pass the blood brain barrier, but
  dopamine is not able to pass the BBB
• Carbidopa is an inhibitor of aromatic amino acid
  decarboxylase in the periphery carbidopa does
  not enter the CNS

35
Serotonin Metabolism (Fig. 19-19)
36
NOS (Fig. 19-23)
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Recycling of Synaptic Vesicles
38
Selected Synaptic Proteins

• Synapsin
• A vesicle protein
• Recruits vesicles to the synaptic region
• Binds to the cytoskelton
• Phosphorylation by PKA and CaM Kinase II releases
  synapsin from vesicles and allows them to move
  into the active region
• v-SNARES for vesicle-(Soluble NSF Attachment
  protein REceptors) and NSF refers to
  N-ethylmaleimide Sensitive Factor
• VAMP vesicle associated protein
• Also called synaptobrevin
• t-SNARES for target
• Syntaxin
• SNAP25 (synaptosomal associated protein MW 25 kDa)

39
Selected Synaptic Proteins II

• Synaptotagmin the calcium ion sensor
• Exocytosis is triggered by Ca2
• Rab3A is a G protein found on vesicles and is
  required for fusion with the plasma membrane and
  exocytosis
• Formation of a VAMP-syntaxin-SNAP25 complex
  occurs with vesicle fusion and exocytosis
• NSF (N-ethylmaleimide sensitive factor), alpha-
  beta-, and gamma-SNAP dissociate the
  VAMP-syntaxin-SNAP25 complex (ATP dependent)
  after fusion
• The proteins return to their initial state (in
  the vesicle or on the target membrane)
• Action potential opens Ca2 channels in the
  synaptic region which triggers exocytosis

40
Vesicle Docking and Fusion
41
Excitation and Inhibition

• Top frog skeletal muscle
• Bottom frog heart
• The Loewi experiment provided proof that
  neurotransmission is chemical in nature (as
  opposed to electrical)
• Vagusstuff (ACh)
• Accelerinstuff (NE)
• Learn this experiment

42
Neurotransmitter Receptors

• Ligand-gated receptors are fast GPCRs are slow
• ACh and the nicotinic receptor at the
  neuromuscular junction is ligand gated and
  promotes the flux of both sodium and potassium
• Nicotinic receptor and other ligand-gated
  receptors consists of 5 subunits
• There are four candidate membrane-spanning
  regions for each subunit
• An M2 alpha helix lines the ion channel
• NT binding triggers a conformational change
  leading to channel opening
• Glutamate
• NMDA, AMPA, and kainate receptors are ionotropic
• The receptor is made of five subunits
• Segments 1,3, and 4 of each are transmembrane
  segments
• Segment 2 courses into, but not through ,the
  membrane from the cytosolic face
• Activation of NMDA requires depolarization and
  glutamate binding
• There are three classes of metabolotropic
  glutamate receptors (7 TM)
• GABA and glycine receptors are ligand-gated Cl-
  channels
• Five subunits per receptor
• Intricate
• Four candidate transmembrane segments

43
Neurotransmitter Receptors II

• ACh and muscarinic receptors in heart
• Causes dissociation of a heterotrimeric G protein
• G beta, gamma binds to and opens a K channel,
  and this leads to hyperpolarization (inhibition)
• G-protein coupled catecholamine receptors lead to
  elevated cAMP

44
Ligand-gated Ion Channel Receptors

• Note that Cl- is responsible for
  hyperpolarization
• Note that Na is responsible for depolarization
• These receptors are made up of 5 subunits each
  with 4 TM segments 5X4 20 TM segments

45
Neurotransmitter Receptors
46
Nicotinic Receptor and the nm Junction

• The formation of autoantibodies against this
  receptor produces myasthenia gravis
• Myasthenia gravis (MG) is a chronic neuromuscular
  disease characterized by varying degrees of
  weakness of the skeletal or voluntary muscles of
  the body
• The muscle weakness increases during periods of
  activity and improves after periods of rest.
• MG most commonly occurs in young adult women and
  older men but can occur at any age
• Although MG may affect any voluntary muscle,
  certain muscles including those that control eye
  movements, eye lids, chewing, swallowing,
  coughing, and facial expressions are more often
  affected
• Weakness may also occur in the muscles that
  control breathing and arm and leg movements.
• Therapies include medications such as
  anticholinesterase agents, prednisone,
  cyclosporine, and azathioprine
• Thymectomy
• Plasmapheresis, a procedure in which antibodies
  are removed from blood plasma

47
Nicotinic ACh Receptor

• Most of the protein mass is extracellular
• There are two acetylcholine binding sites
• There are four membrane TM segments (M1, M2, M3,
  M4) in each of the five subunits (5X420)
• Five M2 helices form the pore
• Aspartate and glutamate side chains at both ends
  of the pore exclude anions

48
Pore-lining M2 Helices

• Closed state kink in the center of each M2 helix
  constricts the passageway
• Open state kinks rotate to one side so that
  helices are farther apart
• Only 3 of the 5 M2 helices are shown

49
Nicotinic Receptor and the nm Junction(Fig.
19-18)
50
NMDA and Non-NMDA Glu Receptors

• NMDA is blocked by Mg2
• Depolarization of several non-NMDA receptors
  leads to depolarization and removal of Mg2
• Ca2 as well as Na traverse the NMDA receptor
• This leads to an enhanced response in the
  postsynaptic cells
• This is long-term potentiation that results from
  a burst of stimulation

51
ACh-induced Opening of K Channels in Heart

• ACh leads to activation of the muscarinic
  receptor
• This leads to the exchange of GTP for GDP in the
  heterotrimeric G-protein
• The beta-gamma subunits activate a K channel
• The outward flow of K leads to a more negative
  intracellular potential, or hyperpolarization,
  and a decreased rate of contraction

52
G-Protein Coupled Receptors (GPCRs)
53
G-Protein Linked Receptors
54
G-Protein Cycle
55
Actions of Heterotrimeric G-proteins

• Stimulate adenylyl cyclase Gs
• Inhibit adenylyl cyclase Gi
• Activate phospholipase C leading to IP3 and
  diacylglycerol production Gq

56
Inactivation of NTs

• Uptake (most prevalent form)
• DA, NE, EPI
• 5-HT
• Glu
• Gly
• Almost all NTs except ACh and neuropeptides
• Julius Axelrod at the NIH discovered
  norepinephrine reuptake and transformed the field
• Hydrolysis
• Neuropeptides
• ACh

57
GABA Metabolism
58
An Electric Synapse

• The plasma membranes of the pre-and post-synaptic
  cells are linked by gap junctions
• Flow of ions through these channels allows
  electric impulses to be transmitted directly from
  one cell to the next
• Unusual in mammals
• Occur in fish (goldfish)

59
Transmission Across Electric and Chemical Synapses

• Transmission across an electrical synapse is fast
  (microseconds)
• Transmission across a chemical synapse occurs on
  the order of milliseconds
• This was the evidence that convinced everyone
  that neurotransmission in the CNS is chemical and
  not electrical in nature

60
Sensory Transduction

• Converts signals from the environment into
  electric signals
• Light G-protein
• Odor G-protein
• Taste gated
• Sound gated
• Touch gated
• Vision
• Stimulated rhodopsin activates transducin, a
  G-protein
• Transducin alpha-GTP activates PDE
• PDE lowers cGMP
• cGMP-gated Na/Ca2 are closed, membrane
  hyperpolarization occurs, and less NT is released
• Each sensory neuron in the olfactory epithelium
  expresses a single type of odorant receptor
• Golf are coupled to and activates adenylyl
  cyclase
• cAMP opens gated channels causing depolarization
  of the cell membrane and generation of an action
  potential
• The thousand or so olfactory receptors are
  intronless

61
Rod Cell
62
Hyperpolarization of the Rod-Cell Membrane

• This system works backwards
• Light causes hyperpolarization and decreased
  released of a NT (Glutamate)

63
Rhodopsin Metabolism
64
Actions in the Rod Cell

• In the dark, the rod cell is hyperpolarized owing
  to the activation of a sodium channel by cGMP
• Light activates rhodopsin, a 7 transmembrane
  segment light receptor (the first 7 TM domain
  protein to be described)
• The heterotrimeric G-protein becomes activated
• The active a-subunit of the G-protein binds to
  the g-subunit of phosphodiesterase (abg) to form
  a complex
• The abcomplex of PDI is now active
• cGMP levels fall, the sodium channel is closed,
  and the cell becomes less depolarized (i.e., more
  polarized or hyperpolarized, and less Glu is
  released)

65
Role of Transducin (Fig. 19-26)
66
Color Vision and Spectra

• Color vision uses three opsin pigments opsins
  are proteins
• These correspond to the three classes of cones
• Blue
• Green
• Red
• Opsins differ, but the pigment is the same
• Red and green opsins are on chromosome X
• Owing to recombination, X chromosomes with only a
  red or a green opsin gene is formed
• 8 of human males leads to red-green blindness

67
Olfactory Epithelium

• The human olfactory epithelium expresses about
  1000 different odorant receptors
• These are G-protein linked
• Golf
• Activate adenylyl cyclase
• cAMP-gated channel induces depolarization
• (b,c) Odorant cells expressing the same receptor
  project to the same point in the olfactory bulb

68
Channel Summary

• Resting, always open
• Voltage gated K and cyclic nucleotide gated
• Four proteins with 6 TM segments 24 TM segments
• Voltage-gated Na and Ca channels
• 24 TM segments
• Ligand gated (ACh, Glu) Na channels
• Five subunits with four TM segments 20 TM
  segments total
• Ligand gated (GABA, Gly)
• Five subunits with four TM segments 20 TM
  segments total