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