13. Neuromuscular Transmission

What controls voluntary muscle contractions?

Motor neurons control contraction

• Primary motor cortex coordinates all voluntary contractions

• Signals travel via upper and lower motor neurons

  • Upper motor neurons synapse with other motor neurons

  • Lower motor neurons synapse with the muscle

• Final outcome → muscle contraction

What are the two types of synapses and how do they differ?

1. Chemical synapses

• Found between neurons or at the NMJ

• Neurotransmitters released from presynaptic neuron → bind postsynaptic receptors

• Signal transduction: electrochemical → chemical → electrochemical

• Slower due to chemical step

2. Electrical synapses

• Rare, mostly in the brain

• Direct flow of electrical current through gap junctions

• Faster, no signal conversion required

How does neuronal signalling achieve convergence?

Convergence = multiple presynaptic inputs integrated by a postsynaptic neuron

• Occurs via neuronal pools

• Brain has >1 billion neurons and >1 trillion synapses, allowing massive integration

What are the two modes of chemical transmission and how do they differ?

1. Fast transmission (ionotropic)

• Neurotransmitter binds ligand-gated ion channels → channel opens directly

• Rapid response

• Example: increase in Ca²⁺ triggers vesicle release at synapse

• Receptor = channel

2. Slow transmission (metabotropic)

• Neurotransmitter binds G-protein-coupled receptor → activates intracellular signaling → ion channels and other processes affected

• Relies on secondary messengers

• Slower, indirect effect

Note:

• Single neurons can use both modes

• Single neurotransmitter can act both ways

How do chemical synapses function at the neuromuscular junction (NMJ)?

Presynaptic terminal contains vesicles storing neurotransmitter (ACh)

• Action potential arrives → vesicles release ACh into synaptic cleft

• Postsynaptic receptors bind ACh → ion channels open → change in membrane permeability → depolarization

• Acetylcholinesterase breaks down ACh → prevents sustained signal

How does acetylcholine act via fast transmission?

Receptor: Nicotinic ACh receptor (nAChR)

• Transmembrane protein complex, 5 subunits (2α subunits bind 1 ACh each)

• Location: NMJ & postganglionic cells of vertebrate autonomic NS

• Mechanism: ACh binding → direct opening of ligand-gated ion channels → rapid depolarization

How does acetylcholine act via slow transmission?

Receptor: Muscarinic ACh receptor (mAChR) → G-protein-coupled receptor (GPCR)

• Mechanism: ACh binds → GPCR activates G-protein on cytoplasmic side → indirectly opens ion channels (via second messengers like cAMP)

• Location: target cells of parasympathetic NS (e.g., cardiac tissue) in vertebrates

• Effect: slower, indirect modulation of postsynaptic cell activity

What happens at the NMJ when an action potential arrives?

Presynaptic AP → release millions of ACh molecules into synaptic cleft

• Muscle depolarizes → opens voltage-gated Na⁺ & K⁺ channels → postsynaptic action potential

• 1 neuron → 1 muscle fiber → organized contraction

• Motor end plate = postsynaptic region of NMJ with high density of nAChRs; only here

• Outside motor end plate: mostly voltage-gated Na⁺ & K⁺ channels, like in neurons

• Graded end plate potential (EPP) → triggers AP; more ACh → more channels open → larger EPP → stronger depolarization

What is the End Plate Potential (EPP)?

• ACh binds to AChR → opens ligand-gated channels → muscle depolarization

• Depolarization opens nearby voltage-gated Na⁺ channels

• If threshold reached → muscle action potential

How do nicotinic ACh receptors function in skeletal vs. cardiac muscle?

Skeletal muscle

• nAChR = non-selective cation channel (permeable to Na⁺ & K⁺)

• ΔgNa / ΔgK ≈ 1.4 → more Na⁺ influx → depolarization

• Mediated by sympathetic NS

Cardiac muscle

• ACh binds muscarinic (metabotropic) receptor

• ↑ gK efflux → hyperpolarization → inhibits contractility

• Mediated by parasympathetic NS

What is the structure of the nicotinic ACh receptor?

ONLY TESTABLE PART: 2α subunits actively bind ACh, permeable to sodium and potassium (higher for sodium)

NOT-TESTABLE:

5 subunits: 2α, 1β, 1γ, 1δ at skeletal NMJ

• Each subunit crosses membrane 4 times (M1–M4)

• Openings at either end are large ~2-3nm in diameter, narrowest region ~-0.6nm (compare this to Na+ and K+ ion which are <0.3nm)

• Central pore formed by 5 subunits, M2 (blue region) important for ion translocation

How does the nAChR select for cations?

M2 region rings contain negatively charged amino acids at the mouth of the channel (inside & outside)
→ These attract and concentrate positively charged cations (Na⁺, K⁺)

• Pore lining: uncharged polar amino acids (e.g., -OH, -NH₂ groups)
  → Provide a selective path for cations to pass through

• Overall mechanism: combination of electrostatic attraction at channel entrances and polar lining inside pore ensures cation selectivity

How can we measure nAChR activation?

Outside-out patch clamp: electrode in ACh solution, measures current through single receptor → shows ligand-gated channel opens

What is the on-cell patch configuration and why is it used to measure nAChR activity?

On-cell patch clamp: measures receptor activity in intact cell membrane

• Limitation: cannot dynamically change ACh concentration in pipette → typically use standard ACh concentration

• Advantage: more physiologically relevant, preserves cytosolic environment and secondary signaling molecules, important for studying metabotropic receptors like muscarinic AChRs

How are macroscopic currents recorded in a muscle fiber, and what does the End Plate Current (EPC) represent?

Individual nACh receptors open briefly when activated by ACh

• Using a voltage clamp, the activity of many receptors at the synapse can be measured

• In skeletal muscle, the brief openings are synchronized → produce a macroscopic current

• This summed current is called the End Plate Current (EPC)

• EPC reflects the influx of many positive ions (Na⁺) through nACh receptors, leading to postsynaptic depolarization

What is the End Plate Potential (EPP) and how does it lead to muscle contraction?

EPP = depolarization of muscle membrane ~+30–40 mV
→ This is threshold for muscle action potential

• Mechanism: inward positive current through nACh receptors depolarizes postsynaptic membrane

• EPP ≠ action potential; it triggers AP by opening voltage-gated Na⁺ & K⁺ channels along the muscle fiber

• Result: suprathreshold depolarization → muscle action potential → contraction

How can we identify which ions flow during the EPC?

Voltage clamp technique is used:

1. Voltage clamp the muscle fiber using 2 electrodes

2. Simultaneously stimulate the presynaptic motor neuron → ACh release

3. Record macroscopic EPCs in response to ACh

• This allows determination of ion fluxes (Na⁺, K⁺) through nACh receptors at the NMJ

How does membrane potential affect the End Plate Current (EPC) in skeletal muscle, and what is the reversal potential?

EPC amplitude & polarity depend on membrane potential (Em):

• Em more negative than resting (-90 mV) → larger inward EPC (stronger Na⁺ influx)

• Em more positive than resting → smaller EPC; at very positive potentials → outward current (K⁺ efflux dominates)

• Reversal potential (ER) = Em where net current = 0

  • For nAChR, ER ≈ 0 mV

  • At ER, EPC reverses direction (inward → outward)

• Key point: ER indicates channel ion selectivity; current direction depends on Na⁺ and K⁺ driving forces

How can we determine which ions flow during the End Plate Current (EPC) through nicotinic ACh receptors?

nAChR channels are permeable to both Na⁺ and K⁺

• Reversal potential (ER) is between ENa and EK, not equal to either

• Changing membrane potential (Em) alters Na⁺ & K⁺ flux → affects amplitude & polarity of EPC/EPP

  • Em = EK → K⁺ has no driving force → EPC is inward Na⁺ current

  • Em = 0 mV (ER) → Na⁺ flux = K⁺ flux → no net current

  • Em = ENa → Na⁺ has no driving force → EPC is outward K⁺ current

• Resting muscle potential: Vm ≈ -90 mV → inward Na⁺ dominates EPC

What are common probes for studying muscle nicotinic ACh receptors and how do they work?

• Bungarotoxin

  • From banded krait snake (Bungarus multicinctus), 74-amino acid toxin

  • Irreversibly binds nAChRs → blocks neurotransmission

  • Competitive inhibitor

  • Used for initial purification of nACh receptor protein

• Curare

  • Plant toxin from Chondrodendron tomentosum

  • Blocks nAChRs via competitive inhibition

What is myasthenia gravis and how does it affect the neuromuscular junction?

Chronic neuromuscular disease → causes skeletal muscle weakness

• Mechanism: autoantibodies attack nicotinic ACh receptors (nAChRs)

• Result: impaired neuromuscular transmission → reduced muscle contraction

How is myasthenia gravis treated pharmacologically?

Strategy: prolong ACh action at NMJ by reducing its breakdown

• Drug example: Neostigmine → inhibits acetylcholinesterase

• Result: more ACh available at nAChRs → improves muscle contraction