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Transcript
receptor G protein i q s t effector channel enzyme intracellular messenger Ca2+ cAMP kinase Bi / CNS 150 Lecture 12 Friday, October 27, 2014 phosphorylated protein The G Protein Pathway in Neuroscience Bruce Cohen Kandel Chapter 11 (Alberts Chapter 15) 1 From a previous lecture Early evidence for chemical synaptic transmission, 1921. Many details of the G protein pathway were first worked out for neuronal control of the heart Vagus nerve runs from the head to the heart Spontaneous heartbeats in both hearts are stopped by stimuli to the “upstream” vagus The diffusible substance: acetylcholine acting on muscarinic ACh receptors smoked drum 2 Several neurotransmitters are agonists for both ligand-gated channels & GPCRs in vertebrates Transmitter Ligand-gated channel GPCR ACh nicotinic AChR muscarinic AChR GABA GABAA GABAB glutamate iGluR mGluR serotonin 5-HT3 5-HTn, n = 1,2, 4-7 histamine (invertebrates only) Hn dopamine (invertebrates only) Dn 3 Plasma Membrane Components of the G Protein Pathway How fast? 100 ms to 10 s How far? Probably less 1 mm Neurotransmitter or hormone binds to receptor activates G protein Effector: enzyme or channel outside a Rasmussen et al., Nature 2011 PDB file 3SN6 GTP b g a GDP + Pi b g inside G protein-coupled receptors receptor G protein i q s t 1. All have 7 a-helices effector channel enzyme 2. There are about 1000 G protein-coupled receptors in the genome. (Most are still “orphans”; their ligands are unknown) intracellular messenger Ca2+ cAMP 3. Individual receptors respond to: a. low-molecular weight neurotransmitter such as serotonin, dopamine, or acetylcholine b. a short protein (8-40 amino acids, a “peptide”) such as an endorphin c. a relatively insoluble lipid such as anandamide, the endocannabinoid d. an olfactory stimulus or, e. light, in the eye (receptor = rhodopsin) 5 Structure of a heterotrimeric G protein: a molecular switch receptor β subunit G protein i q s t effector α subunit intracellular messenger GDP γ subunit PDF file: 1GOT Note the “propeller” in the b subunit which caps the a subunit, preventing either subunit from interacting with the effector (There is no effector in this structure): Three types of G proteins are involved in neurotransmitter signal transduction • Gi proteins are typically involved in neuronal inhibition (hence the “i” subscript) • Gq proteins bind to enzymes that ultimately induce Ca2+ release into the cytoplasm • Gs proteins bind to enzymes that catalyze the synthesis of intracellular messengers such as cAMP and cGMP 7 Gi-coupled receptors usually inhibit neurons Gi directly activates some K channels Gi directly inhibits some voltage-gated Ca channels Gi directly inhibits adenylyl cyclase All these actions slow neuronal firing and decrease transmitter release 8 receptor G protein-gated K channels inhibit neuronal (& cardiac) firing Resting GK EK -90 mV effector channel enzyme Voltage-gated Ligand-gated G protein-gated outside GEPSP EEPSP ~ -5 mV G protein i q s t GCl ECl -80 mV GNa GK GK ENa +50 mV EK -90 mV EK -90 mV intracellular messenger Ca2+ cAMP Capacitance cytosol = inside +60 additional K+ channels keep the membrane potential away from threshold, and therefore decrease firing rates mV -60 1 ms 5 G protein gated K+ channels (GIRKs) are inward rectifiers. When activated, they “latch” the cell quiet until excitatory stimuli finally succeed in depolarizing to threshold. E K GK + E EPSP GEPSP + E Cl GCl + E NaGNa V= GK + G EPSP + G Cl + G Na receptor Gq, Gs, and Gt protein effectors include some enzymes Gq G protein i q s t effector channel enzyme Enzyme Ca2+ in endoplasmic reticulum Ca2+ in cytosol intracellular messenger Ca2+ cAMP 10 phosphatidyl inositol 4,5 bisphosphate = PI(4,5)P2 An example of an intracellular, ligand-gated channel Alberts et al., Molecular Biology of the Cell, © Garland Science KCNQ channels PIP2 is necessary for keeping some K channels open. Gq activation leads to less PIP2 Result: some K channels close. These are called “M” channels, and are now termed the KCNQ family. because they were first discovered downstream from muscarinic receptors . . . A different muscarinic receptor subtype from the one that opens K channels in heart. Figure 11-11 12 receptor G protein i q s t Gq, Gs, and Gt protein effectors include some enzymes: effector channel enzyme Gs-coupled receptors often stimulate neurons & other cells intracellular messenger Ca2+ cAMP ATP 2+ Mg2+ Mg NH 2 N N O O O -O P O P O P O CH 2 O H H OOOH OH OH ATP cyclic AMP (cAMP) Gs NH 2 N N N N N cyclase O O O P -O O N H H OH cyclic AMP (cAMP) See Figure 11-3 13 cAMP binds to kinase; this activates the kinase. intracellular messenger Ca2+ cAMP kinase phosphorylated protein Alberts 11-31 © Garland serine O N CHC O H CH2 OH Residue in target protein kinase phosphatase O N CHC O H CH2 O -O P O O 14 Example of ion channel phosphorylation: β-adrenergic receptors regulate accommodation in hippocampal neurons intracellular messenger Ca2+ cAMP kinase Apply norepinephrine phosphorylated protein Norepinephrine inhibits the SK (smallconductance, Ca2+ -activated K+) channel. Apply 8-bromo-cAMP Apply forskolin (then apply glutamate in the presence of TTX) epsp Therefore the after-hyperpolarization (AHP) is smaller and spike trains are longer. The norepinephrine effect is also mimicked by agents that mimic or increase cAMP. 1. phosphodiesterase.does not hydrolyze 8-bromo-cAMP 2. Forskolin activates cyclase intracellular messenger Ca2+ cAMP kinase phosphorylated protein kinase phosphorylated protein Nucleus 16 Many genes have a DNA sequence called “cAMP-Ca2+ responsive element” (CRE) intracellular messenger Ca2+ cAMP kinase Target or reporter gene CRE pCREB phosphorylated protein The transcription factor that binds to this CRE: “cAMP-Ca2+ responsive element binder” (CREB). O -O P O O Alberts et al., Molecular Biology of the Cell, © Garland Science from Lecture 12 outside receptor membrane b g G protein i q s t a b g a inside effector channel enzyme The pathway from GPCR to gene activation intracellular messenger Ca2+ cAMP cytosol kinase phosphorylated protein nucleus How fast? 10 s to days How far? Up to 1 m 18 End of Lecture 12 19