Week 13
Describe active zones.
Double linear array of synaptic vesicles and intramembranous particles Oriented directly over secondary postsynaptic clefts that lie between adjacent post junctional folds
Where are the N2 AChRs found?
Autonomic nervous system on the postsynaptic membrane of the postganglionic sympathetic and parasympathetic neurons And also in the CNS
What is the synaptic basal lamina?
A particular region of the muscle basement membrane called the synaptic basal lamina contains various proteins that mediate adhesion of the NMJ and play important roles in synapse development and regeneration.
What are the agonists of the N1 nAChR?
ACh (nicotine, decamethonium)
What are the agonists of N2 nAChRs?
ACh (nicotine, tetramethylammonium)
How is an excitatory end-plate current produced?
ACh activates nicotinic AChRs to produce an excitatory end plate current. Transient depolarization in the muscle membrane after delay of few milliseconds. This delay represents the time required for the release of ACh, its diffusion across the synapse, and activation of post synaptic AChRs. The positive voltage change follows a biphasic time course This is known as an end plate potential, and is an example of an EPSP
Describe the synthesis of ACh
ACh is synthesized in the nerve terminal, outside the vesicle, from choline and acetyl coA by the enzyme choline acetyltransferase
How do noncompetitive agonists inhibit the work of the nAChR?
Act by entering the lumen of the channel and blocking the flow of ion current.
What does a curare blockade do?
Allows only a small number of AChR channels to open, so that the EPP does not reach threshold to produce an AP.
Contrast the ion permeability and current produced by opening of serotonin receptors, glycine receptors, and GABAa receptors.
As is the case for AChRs, the 5-HT3 receptor channels are permeable to cations and thus produce excitatory currents. In contrast, glycine-activated and GABAa channels are permeable to anions such as Cl- and produce inhibitory currents.
Where are the N1 AChRs located?
At the neuromuscular junction
Where is the reversal potential for the EPC?
Close to 0 mV Because the EPC specifically corresponds to current thorough AChR channels, this reversal potential reflects the ionic selectivity of these channels when extracellular sodium and potassium concentrations are normal
Describe the subunit structure of the nicotinic AChR.
Consists of four subunits (a, B, y, d) in a pentameric (heteropentamer) fashion (2a:1B:1Y:1d) The subunits each have four distinct hydrophobic regions known as M1 to M4 which correspond to membrane spanning segments The M2 transmembrane segment lines the aqueous pore through which Na+ and K+ cross the membrane The pentameric complex has two agonist binding sites. These are located at the extracellular a/y interface of one a subunit and the a/d interface of the other a subunit Because the five subunits are not identical, the structure exhibits pseudo symmetry, rather than true symmetry
What is the basis for differences in nicotinic AChRs?
Different subunit composition
Where does the process of fusion of synaptic vesicles and release of ACh occur?
Differentiated regions of the presynaptic membrane called active zones.
Describe nonjunctional AChRs
Fetal receptors Pentameric complex with a subunit composition of a2Byd
Why, after the addition of QX-222, does the channel exhibit a rapidly flickering behavior?
Flickering represents a series of brief interruptions of the open state by numerous closures. This type of flickering block is caused by rapid binding and unbinding of the drug to a site in the mouth of the open channel. When the drug binds, it blocks the channel to the flow of ions (A2B) Conversely, when the drug dissociates, the channel becomes unblocked (A2O)
What is the antagonist of N2 AChRs?
Hexamethonium
Conclude why recording in curare treated muscle at various distances from the end plate, the amplitude of the potential change is successively diminished and its peak is increasingly delayed.
In this experiment, curare blockade allows only a small number of AChR channels to open, so that the EPP does not reach the threshold to produce an AP. If the experiment is repeated by inserting the micro electrode at various distances from the end plate, the amplitude of the potential change is successively diminished and its peak is increasingly delayed. This decrement with distance occurs because the EPP originates at the end-plate region and spreads away from this site according to passive cable properties of the muscle fiber. Thus, the EPP is an example of a propagated graded response.
What is the purpose of post junctional folds?
Increase the surface area of the muscle plasma membrane in the postsynaptic region
What type of receptors are the N1 and N2?
Ligand-gated ion channels activated by ACh or nicotine
Describe the NMJ.
Motor neurons with cell bodies located in the ventral horn of the spinal cord have long axons that branch extensively near the point of contact with the target muscle. Each axon process innervates a separate fiber of skeletal muscle
What are the two major subtypes of nAChRs?
N1 and N2
What are terminal arborizations?
Small tree-like patch of unmyelinated nerve processes found on each individual end plate.
What is an end plate?
Neuromuscular junction: specialized synapse region where a skeletal muscle and axon meet
Why does nicotinic stimulation lead to rapid depolarization?
Permeable to both Na+ and K+, but Na+ dominates, therefore leading to rapid depolarization
What does a competitive antagonist such as d-tubocurarine do to the binding of ACh?
Prevents the binding of the agonist ACh to each of its two sites
Describe the activation of AChR channels
Requires binding of two ACh molecules. The closed state of the channel must bind one molecule of ACh to form a closed-agonist-bound channel (AC) before it can convert to an open-agonist-bound channel (AO). C --> A1C --> A2C --> A2O
What are shwann cells?
Shwann cells are intimately associated with the nerve terminal and forma cap over the face of the nerve membrane that is located away from the muscle membrane.
When activated, N1 and N2 receptors are both permeable to...
Sodium and potassium Entry of sodium dominates Therefore the nicotinic stimulation leads to rapid depolarization
Describe how ACh moves into the synaptic vesicle.
The ACh moves into the synaptic vesicle via a specific ACh-H exchanger, which couples the inward transport of ACh to the efflux of H+.
Describe the characteristic time course of the end plate current
The EPC has a characteristic time course that rises to a peak within 2 ms after stimulation of the motor nerve and falls exponentially back to zero. The time course of the EPC corresponds to the opening and closing of a population of AChR channels governed by the rapid binding and disappearance of ACh as it diffuses to the postsynaptic membrane and is hydrolyzed by AChE.
Describe the different subunit conformations of N1 nAChRs.
The N1 receptors have different subunit compositions depending upon location and developmental stage. Fetal skeletal muscle and nonjunctional regions of denervated adult skeletal muscle: a2Byd NMJ of adult skeletal muscle: a2Bed
Describe the subunit composition of N2 nAChRs
The N2 receptors are heteromers, probably heteropentomers. N2 receptors use a2 to a10 and b2 to b4
What happens if the experiment is repeated by inserting the micro electrode at various distances from the end plate?
The amplitude of the potential change is successively diminished and its peak is increasingly delayed This decrement with distance occurs because the EPP originates at the end-plate region and spreads away from the site according to passive cable properties of the muscle fiber Thus, the EPP left is an example of a propagated graded response.
What are boutons?
The bulb-shaped endings that contact the muscular fiber in an NMJ
Molecular localization studies have shown that the density of ionotropic (nicotinic) AChRs is very high at...
The crests of post junctional folds
The synaptic basal lamina contains a high concentration of...
The enzyme acetylcholinesterase (AChE) Ultimately terminates synaptic transmission by rapidly hydrolyzing free ACh to choline and acetate.
Explain why, after the addition of QX-222, an analog of the local anesthetic agent lidocaine, to the extracellular side, the channel exhibits a rapidly flickering behavior.
The flickering represents a series of brief interruptions of the open state by numerous closures. This type of flickering block is caused by rapid binding and unbinding of the anesthetic drug to a site in the mouth of the open channel. When the drug binds, it blocks the channel to the flow of ions. Conversely, when the drug dissociates, the channel becomes unblocked.
What is inside of the synaptic cleft?
The intervening space of the synaptic cleft is filled with a meshwork of proteins and proteoglycans that are part of the extracellular matrix
Describe the pentameric cos-loop receptor family of ligand-gated ion channels
The nAChRs belong to this family because they contain a highly conserved pair of disulfide-bonded cysteine residues. This family also contains three other classes of agonist-activated channels: serotonin, glycine, and GABA Serotonin is cation permeable and glycine and GABA are anion permeable
What happens when the nicotinic AChR channel at the muscle end plate opens?
The normally high resting permeability of the muscle plasma membrane for K+ relative to Na+ falls so that Na+ and K+ become equally permeant and Vm shifts to a value between Ek and Ena
What are post junctional folds?
The postsynaptic membrane of the skeletal muscle fiber lying directly under the nerve terminal is characterized by extensive invaginations known as post junctional folds.
Explain in detail why the reversal potential for the nicotinic AChR channel is close to 0 mV.
The reversal potential is near 0 mV because the nAChR has a poor selectivity for Na+ vs K+. When the nAChR channel at the muscle end plate opens, the normally high resting permeability of the muscle plasma membrane for K+ relative to Na+ falls so that Na+ and K+ become equally permeant and Vm shifts to a value between Ek and Ena.
What enzyme is present in high concentrations at the basal lamina and what is its role in synaptic transmission?
The synaptic basal lamina contains a high concentration of the enzyme acetylcholinesterase (AChE) which ultimately terminates synaptic transmission by rapidly hydrolyzing free ACh to choline and acetate.
What are the different functional properties of junctional and nonjunctional AChRs and what is responsible for them?
The unitary conductance of nonjunctional receptors is smaller and the single channel lifetime is longer in duration than that of junctional receptors. measurements of currents at different voltages yielded single-channel IV curves showing that the channel formed with the E subunit had a unitary conductance of 59 pS, whereas that formed with the Y subunit had a conductance of 40 pS. The mean lifetime of single channel openings at 0 mV was 1.6 ms for E type and 4.4 ms for Y type receptors, which closely corresponds to values found in adult muscle and native fetal, respectively. The basis for this phenomenon is a difference in subunit composition. The nonjunctional (fetal) receptors are a pentameric complex with a subunit composition of a2BYd. For the junctional AChR (adult skeletal muscle) substitution of an E subunit for the fetal Y subunit results in a complex with the composition a2BEd
Describe the differences in unitary conductance of nonjunctional receptors vs junctional receptors.
The unitary conductance of nonjunctional receptors is smaller and the single-channel lifetime is longer in duration than that of junctional receptors. The basis for this phenomenon is difference in subunit composition
What is a motor unit?
The whole assembly of muscle fibers innervated by the axon from one motor neuron
How is the EPP produced?
Transient opening of AChR channels, which are selectively permeable to monovalent cations such as sodium and potassium. The increase in sodium conductance drives the membrane potential to a more positive value in the vicinity of the end plate region
What is the process of ACh --> synaptic vesicle energetically driven by?
Vesicular proton electrochemical gradient (positive voltage and low pH inside) which in turn is produced by a vacuolar-type H pump fueled by ATP. The nerve terminal also contains numerous mitochondria that produce the ATP required to fuel energy metabolism.
What happens when the muscle fiber is clamped to a holding potential of -120mV
We observe a large inward current (i.e. the EPC) This inward current decreases in magnitude as the membrane potential is made more positive, and the current reverses direction to become an outward current at positive values of Vm.
Is the selectivity of AChR channel weak or strong?
Weak ionic selectivity This selectivity of the AChR is well suited to its basic function of raising Vm above the threshold of about -50mV, which is necessary for the firing of an AP
Describe junctional AChRs.
adult receptors in skeletal muscles a2bed
What is the neuromuscular junction?
connection between the motor neuron and the muscle fiber Specialized synapse between motor neuron and skeletal muscle
What are the antagonists of the N1 nAChR?
d-tubocuranine and a-bungarotoxin
The receptor-gated channels for serotonin and glutamate are cation selective and give rise to...
depolarizing excitatory postsynaptic potentials
The receptor-gated channels for glycine and GABA are anion selective and...
drive Vm in the hyper polarizing direction, toward the equilibrium potential for Cl-. These hyper polarizing postsynaptic responses are called inhibitory postsynaptic potentials
What are the two types of AChRs?
junctional and nonjunctional receptors
Where does the basis for cation vs anion selectivity appear in the pentameric cos-loop receptor family?
within the M2 segment
