Human Cellular Physiology PHSI3004/3904

Secreted signals and synaptic transmission

Dr Bill Phillips

Dept of Physiology, Anderson Stuart Bldg Rm N348

Secreted signals and synaptic transmission

• Chemical signalling between cells

• Ca2+ and chemical synaptic transmission

• Neuromuscular synapse

• Quantal Release

• Vesicle exocytosis and fusion pore

• Synaptic vesicle cycle

• Organisation of the release site• Kandel et al.2000 Cpts 11 & 14

Types of chemical signals

Nuc.

Cellular Response

Nuc.

Cellular Response

Nuc.

Nuc.

Cellular Response

water soluble

hydrophobic

Forms of release of hydrophilic signalling chemicals

• Release from the cytoplasm- regulated membrane channels or transporters

• Release from membrane vesicle stores-regulated fusion pore and/or exocytosis

Studying controlled (evoked) neurotransmitter release

Nucleus

Muscle fibre

Experimental evidence for the role of Ca2+ in transmitter release

• Giant synapse of the squid made it possible to study relationship between presynaptic events and neurotransmitter release.

• Intracellular electrodes in the nerve terminal recorded presynaptic membrane potential

• Intracellular electrode in the postsynaptic cell recorded the excitatory postsynaptic potential (a measure of transmitter release)

Ca2+ influx controls transmitter release

• Presynaptic nerve terminal was voltage clamped

• Voltage gated Na+ and K+ channels were blocked

• Step depolarisation used to open voltage-gated Ca2+ channels

• Small increases in inward Ca2+ current led to much bigger proportional increases in postsynaptic response (gauge of transmitter release)

Kandel et al. 2000 Fig 14-3

Relationship between Ca2+ influx and transmitter release

• Transient increase in [Ca2+]i depends upon both [Ca2+]o and conductance (number of voltage-gated Ca2+ channels open

• Two-fold increase in [Ca2+]o results in as much as a 16-fold increase in transmitter release (4-power relationship)

• Implies multiple, low affinity binding sites (as many as 4) on “calcium sensor”

Kandel et al 2000 Fig 14-4

Time course of pre- synaptic Ca2+ influx

Inward Ca2+ current follows the presynaptic AP and precedes the postsynaptic potential as little as 0.2msec

Short delay between Ca2+ influx and transmitter release suggestsCa2+ channels are closely adjacent to Ca2+ sensor and transmitter release site. Ca2+ channels thought to be concentrated in discrete release zones on nerve terminal

Types of voltage-gated Ca2+ channels (1 pore-forming subunits encode primary properties)

Ca2+ channel type Cellular localisation/

functionP/Q Nerve terminals/release

N Nerve terminals/release

R Nerve terminals/release

L nerve,muscle, endocrine

T Neruons,heart/excitability

Neuromuscular Synapse “model”

• Vertebrate neuromuscular synapses display highly regulated neurotransmitter release

• One nerve cell (motor neuron) controls one target cell (muscle fibre) by releasing acetylcholine (ACh) onto cation channels gated by ACh.

• A high density of ACh receptor/channels ensures that the postsynaptic membrane potential responds quickly and quantitatively to the amount of transmitter released by the nerve terminal.

Structure and molecularorganisation: plan view

Postsynaptic acetylcholine receptors Presynaptic nerve terminal

Last internode

Terminal branches

10μm

Miniature endplate potentials

• Intracellular recordings from the postsynaptic membrane of skeletal muscle fibres show occasional small amplitude depolarisations of ~0.5mV lasting ~2msec called miniature endplate potentials MEPP.

• Amplitude of mEPPs decline exponentially with distance from the synapse just like the nerve-evoked endplate potential (EPP)

MEPPs arise from release of quanta of acetylcholine

• Each acetylcholine receptor (AChR) channel can depolarise the membrane by only about 0.3μV

• Thus MEPP (0.5mV) must involve simultaneous opening of ~2,000 AChR channels

• Since the AChR has two AChR binding sites and allowing for loss of ACh in the synaptic cleft, a ‘quantum’ of ~5000 molecules of ACh must be released to generate a MEPP

Recording the EPP

+30mV

0mV

-90mV

Stimulateaction potentials

Record Vm

Evoked release of acetylcholine occurs in multiples of the quantal amount

• When [Ca2+]o is reduced below physiological levels the amplitude of the EPP declines greatly from ~70mV to 0.5- 3mV range, varying from trial to trial

• Frequency distributions show that amplitudes of EPPs fell into multiples of the mean amplitude of the spontaneously occurring MEPP

Kandel et al. 2000 Fig 14-6

Number of quanta released depends upon Ca2+ influx

• Quanta are released spontaneously (MEPPs) but at very low frequency

• Brief high concentration bursts Ca2+ (~0.1mM) massively increases probability of release occuring adjacent to calcium channels

• Neuromuscular synapses contain many release sites so coordinated release of ~150 quanta occur, leading to the normal EPP

Quanta are thought to be contained in and released from synaptic vesicles

• Nerve terminals contain ~200 synaptic vesicles each about 50nm diameter

• These contain neurotransmitter• Electron microscopic rapid freeze evidence

indicates synaptic vesicle exocytosis follows nerve terminal depolarisation

• Membrane capacitance increases in nerve terminals suggest fusion of vesicle membrane with plasma membrane

Fusion pores

• Precise steps in release of transmitter from a synaptic vesicle not fully understood

• First step may be formation of a fusion pore the diameter of a gap junction (~2nm)

• Some transmitter may diffuse out through this pore

• In most cases this is though to dilate to ~8nm leading to full exocytosis

Capacitance evidence for vesicle exocytosis and a fusion pore

Kandel et al. 2000 Fig 14-10

“Kiss and Run” release

• In some situations the 2nm diameter fusion pore seems to open then close again, without fully dilating

• This is known as kiss and run release

• It may simplify and speed up recovery and recycling of the synaptic vesicles

Synaptic vesicle recycling

Kandel et al. 2000 Fig 14-12

Kandel et al 2000 Fig 14-5

Voltage gated Ca 2+ channels are aligned in rows overlying clusters of postsynaptic ACh receptors