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 hydrophobic Nuc.

Nuc.

Cellular Response wat er soluble Nuc.

Cellular Response Nuc.

Cellular Response

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 Muscle f ibre Nucleus

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. 2000 Fig 14-3

Relationship between Ca 2+ and transmitter release influx • Transient increase in [Ca 2+ ] i both [Ca 2+ ] o voltage-gated Ca 2+ depends upon and conductance (number of 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 and transmitter release suggests Ca 2+ 2+ influx 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

P/Q N R L T Types of voltage-gated Ca 2+ channels (  1 pore-forming subunits encode primary properties)

Ca 2+ channel type Cellular localisation/ function

Nerve terminals/release Nerve terminals/release Nerve terminals/release nerve,muscle, endocrine 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 molecular organisation: plan view Postsynaptic acetylcholine receptors Presynaptic nerve termina l 10  m Last internode Terminal branches

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 Stimulate action potentials +30mV 0 mV - 9 0 m V Record V m

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 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 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. 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

Voltage gated Ca in rows overlying ACh receptors 2+ channels are aligned clusters of postsynaptic Kandel et al 2000 Fig 14-5