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

7 transmembrane helices G protein binding domain G protein coupled receptors 6

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... G protein coupled receptors 7

The 7TM/GPCR receptor family 8

9

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 Exposes binding site for G protein G protein binds to receptor GTP replaces GDP 18

G proteins Let’s look at the steps in detail Transmitter binds Exposes binding site for G protein G protein binds to receptor GTP replaces GDP α subunit of G protein separates from βγ subunits Both parts can now activate various effectors 19

G proteins Let’s look at the steps in detail Transmitter binds Exposes binding site for G protein G protein binds to receptor GTP replaces GDP α subunit of G protein separates from βγ subunits Both parts can now activate various effectors α subunit binds to effector (in this case adenylate cyclase) and activates it 20

G proteins Let’s look at the steps in detail Transmitter binds Exposes binding site for G protein G protein binds to receptor GTP replaces GDP α subunit of G protein separates from βγ subunits Both parts can now activate various effectors α subunit binds to effector and activates it Hydrolysis of GTP to GDP leaves the α subunit unable to activate the effector It rejoins with the βγ subunits 21

G proteins Let’s look at the steps in detail Transmitter binds Exposes binding site for G protein G protein binds to receptor GTP replaces GDP α subunit of G protein separates from βγ subunits Both parts can now activate various effectors α subunit binds to effector and activates it Hydrolysis of GTP to GDP leaves the α subunit unable to activate the effector It rejoins with the βγ subunits As long as transmitter remains bound to receptor the G protein can be reactivated Unbinding of transmitter 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 Ca 2+ entry 24

How does noradrenaline work? NA Ca 2+ channel NA applied outside the cell Ca 2+ channel recorded in cell attached patch 25

NA increases Ca 2+ channel activity How does noradrenaline work? 26

NA How can NA outside the cell activate a Ca 2+ channel in the patch? There can’t be a direct connection There must be a mobile “second messenger” inside the cell Ca 2+ channel How does noradrenaline work? 27

How does noradrenaline work? 28

Directly apply activated protein kinase A + ATP Same effect as that of noradrenaline Evidence for role of phosphorylation 29

Noradrenaline effect on the heart Noradrenaline activates β-adrenergic receptor - This activates G protein (G s ) - G s 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 Slow inward current : “pacemaker” depolarisation Reduced pacemaker depolarisation: reduced inward current OR increased outward current (This was the Vagusstoff effect that Loewi studied) 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 ACh Amanita muscaria: source of muscarine 35

Is a soluble second messenger involved? ACh K + channel ACh applied outside the cell K + channel recorded in cell- attached patch No effect! 36

No effect of ACh in bath 37

How does ACh affect K + channels? ACh K + channel 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 G βγ Inside-out patch ACh applied in pipette (extracellular) G βγ 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 (G i ) - G i 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 ) 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 ); chapter 8 (pages ); box 6B (page 117) Nicholls et al chapter 12 (pages ); chapter 22 (pages ) Kandel et al chapter 12, chapter 63 (pages ), chapter 16