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 Ca 2+ 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

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

Experimental evidence for the role of Ca 2+ 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)

Ca 2+ 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 Ca 2+ channels Small increases in inward Ca 2+ current led to much bigger proportional increases in postsynaptic response (gauge of transmitter release) Kandel et al Fig 14-3

Relationship between Ca 2+ influx and transmitter release Transient increase in [Ca 2+ ] i depends upon both [Ca 2+ ] o and conductance (number of voltage-gated Ca 2+ channels open Two-fold increase in [Ca 2+ ] 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 Ca 2+ influx Inward Ca 2+ current follows the presynaptic AP and precedes the postsynaptic potential as little as 0.2msec Short delay between Ca 2+ influx and transmitter release suggests Ca 2+ channels are closely adjacent to Ca 2+ sensor and transmitter release site. Ca 2+ channels thought to be concentrated in discrete release zones on nerve terminal

Types of voltage-gated Ca 2+ channels (  1 pore-forming subunits encode primary properties)

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.

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 0.3  V 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

Evoked release of acetylcholine occurs in multiples of the quantal amount When [Ca 2+ ] o is reduced below physiological levels the amplitude of the EPP declines greatly from ~70mV to mV 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 Fig 14-6

Number of quanta released depends upon Ca 2+ influx Quanta are released spontaneously (MEPPs) but at very low frequency Brief high concentration bursts Ca 2+ (~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 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 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