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Transcript
•Next theme: ion channel modulation (or “indirect” synaptic transmission) 1 Reminder: The postsynaptic membrane 2 Direct and indirect synaptic transmission: where do they happen? Direct: •neuromuscular junction (ACh) •central excitatory synapses (glutamate) •central inhibitory synapses (GABA, glycine) Indirect (today’s theme) •parasympathetic nerve action on heart and other organs (ACh) •sympathetic nerve action on heart and other organs (adrenaline, noradrenaline) •central excitatory and inhibitory synapses (glutamate, GABA, peptide transmitters) 3 Indirect transmission can be complex 4 G protein coupled receptors 5 G protein coupled receptors 7 transmembrane helices G protein binding domain 6 G protein coupled receptors •7 transmembrane helices •G protein binding domain •These characteristics define a very large receptor family: 7TM/GPCR family •About 300 known human sequences for receptors in this family... 7 The 7TM/GPCR receptor family 8 The 7TM/GPCR receptor family 9 The 7TM/GPCR receptor family 10 The 7TM/GPCR receptor family 11 The 7TM/GPCR receptor family ...plus about 300 members of the family which are olfactory receptors •Many others are taste receptors •Rhodopsin and its relatives (the light sensitive proteins from the eye) are also members of this family 12 The 7TM/GPCR receptor family Different binding sites for different types of agonists 13 G proteins •All these receptors bind to and activate G proteins •G proteins are GTP-binding proteins •They “pass on” the message of receptor activation to further processes within the cell 14 G proteins •Activation of the receptor (star) causes G protein (αβγ) to split •Either the α or the βγ parts can activate various further processes 15 G proteins •Let’s look at the steps in detail Adenylate cyclase 16 G proteins •Let’s look at the steps in detail •Transmitter binds •Exposes binding site for G protein 17 G proteins •Let’s look at the steps in detail •Transmitter binds •G protein binds to receptor •Exposes binding site for G protein •GTP replaces GDP 18 G proteins •Let’s look at the steps in detail •Transmitter binds •G subunit •α protein of binds G protein to receptor separates from βγ subunits •Exposes siteactivate for G protein •GTP replaces •Both partsbinding canGDP now various effectors 19 G proteins •Let’s look at the steps in detail •Transmitter •G subunit •α protein binds of binds G protein to effector receptor separates (in thisfrom caseβγ adenylate subunits •Exposes binding siteactivate for •GTP replaces •Both cyclase) parts and can activates GDP now it G protein various effectors 20 G proteins •Let’s look at the steps in detail •Transmitter •G subunit •α •Hydrolysis protein binds ofofbinds GGTP protein to effector receptor to GDP separates and leaves activates from the αβγsubunit itsubunits unable •Exposes siteactivate for G protein •GTP •Both to activate replaces partsbinding the caneffector GDP now various effectors •It rejoins with the βγ subunits 21 G proteins •Let’s look at the steps in detail •Transmitter binds •G subunit •α •Hydrolysis •As protein long asbinds ofof transmitter GGTP protein to effector receptor to GDP separates remains and leaves activates bound from the αβγ tosubunit itreceptor subunits unable the •Exposes binding siteactivate for G protein •GTP •Both to G protein activate replaces parts can the can be effector GDP now reactivated various effectors •It rejoins with •Unbinding of transmitter the βγ subunits ends the sequence 22 Example: making the heart beat stronger 23 Noradrenaline increases force of contraction •Noradrenaline (norepinephrine): transmitter released from sympathetic nerves •Increases heart rate and force of contraction •Force increases due to increased Ca2+ entry 24 How does noradrenaline work? Ca2+ channel NA •NA applied outside the cell •Ca2+ channel recorded in cell attached patch 25 How does noradrenaline work? •NA increases Ca2+ channel activity 26 How does noradrenaline work? Ca2+ channel NA •How can NA outside the cell activate a Ca2+ channel in the patch? •There can’t be a direct connection •There must be a mobile “second messenger” inside the cell 27 How does noradrenaline work? 28 Evidence for role of phosphorylation •Directly apply activated protein kinase A + ATP •Same effect as that of noradrenaline 29 Noradrenaline effect on the heart •Noradrenaline activates β-adrenergic receptor - This activates G protein (Gs) - Gs activates adenylyl cyclase to synthesise cyclic AMP - cAMP activates protein kinase A - Protein kinase A phophorylates calcium channel - Phosphorylation activates the calcium channel •The response is switched off in two ways: - Phosphatases remove the phosphate groups from the ion channels - Phosphodiesterases break down cAMP •All adrenergic receptors are members of the 7TM/GPCR family 30 Example 2: how ACh slows the heart 31 Example 2: how ACh slows the heart (This was the Vagusstoff effect that Loewi studied) Slow inward current : “pacemaker” depolarisation Reduced pacemaker depolarisation: reduced inward current OR increased outward current It turned out to be an increased outward current, i.e. opening of a K+ channel 32 Two types of ACh receptors Nicotinic: activated by nicotine as well as ACh. Neuromuscular junction and CNS Muscarinic: activated by muscarine as well as ACh. Parasympathetic nervous system and CNS 33 Two types of ACh receptors Amanita muscaria: source of muscarine Muscarinic: activated by muscarine as well as ACh. Parasympathetic nervous system and CNS 34 Two types of ACh receptors Muscarine Amanita muscaria: source of muscarine ACh 35 Is a soluble second messenger involved? K+ channel ACh •ACh applied outside the cell •K+ channel recorded in cellattached patch •No effect! 36 No effect of ACh in bath 37 How does ACh affect K+ channels? K+ channel ACh •ACh applied in the pipette •K+ channel now activated by ACh 38 ACh in pipette activates K+ channel 39 How does ACh work? •Membrane-delimited pathway •The βγ subunits of the G protein activate the K+ channel 40 Evidence for role of Gβγ •Inside-out patch •ACh applied in pipette (extracellular) •Gβγ applied in bath (intracellular) •Effect of both is the same 41 ACh effect on the heart •“Membrane delimited” pathway, initially surprising but many examples discovered since •ACh activates muscarinic receptor - This activates G protein (Gi) - Gi splits into α and βγ subunits - The βγ subunits directly activate the K+ channel •The response is switched off by unbinding of the βγ subunits 42 Reading for this lecture: •Purves et al chapter 7 (up to page 153) Nicholls et al chapter 10 (especially pages 184-188) •Kandel et al chapter 13 (Note: All these readings go into a lot of depth, so read selectively based on examples in the lecture) Next lecture Synaptic integration, plasticity and myasthenia gravis •Purves et al chapter 5 (pages 101 - 107); chapter 8 (pages 169-177); box 6B (page 117) •Nicholls et al chapter 12 (pages 232-238); chapter 22 (pages 449-452) •Kandel et al chapter 12, chapter 63 (pages 1259-1272), chapter 16