Biochemistry of Neurotransmission: A Type of

Cell-Cell Signaling

Biochemistry is fun

Biochemistry of Cell Signaling

Fig. 19-1

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?

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

Neuroanatomy

Fig. 19-2

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

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

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)

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

Synapses

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

Ion Channels

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

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

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

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)

Myelinated and Non-Myelinated

Nerves in Dental Pulp

Structure of a Peripheral Myelinated Axon

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

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

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

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

Structure and Function of the Voltage-gated Na+ Channel

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

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

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

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

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

Selected Neurotransmitters

ACh at the vertebrate nm junctionGlutamate at the invertebrate nm junction (crayfish and lobster)

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 (?)

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

• ACh is inactivated by hydrolysis

Acetylcholine Congeners (Fig. 19-17)

Catecholamines

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

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

Serotonin Metabolism (Fig. 19-19)

NOS (Fig. 19-23)

Recycling of Synaptic Vesicles

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)

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

Vesicle Docking and Fusion

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

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

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

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

Neurotransmitter Receptors

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

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 (5X4=20)– Five M2 helices form the pore

• Aspartate and glutamate side chains at both ends of the pore exclude anions

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

Nicotinic Receptor and the

nm Junction(Fig. 19-18)

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

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

G-Protein Coupled Receptors (GPCRs)

G-Protein Linked Receptors

G-Protein Cycle

Actions of Heterotrimeric G-proteins

• Stimulate adenylyl cyclase: Gs

• Inhibit adenylyl cyclase: Gi

• Activate phospholipase C leading to IP3 and diacylglycerol production: Gq

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

GABA Metabolism

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)

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

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

Rod Cell

Hyperpolarization of the Rod-Cell Membrane

• This system works “backwards”

• Light causes hyperpolarization and decreased released of a NT (Glutamate)

Rhodopsin Metabolism

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 -subunit of the G-protein binds to the

subunit of phosphodiesterase () to form a complex• The –complex 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)

Role of Transducin (Fig. 19-26)

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

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

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