Neuropeptides and Slow Neurotransmission PDF

Neuropeptides and Slow Neurotransmission Learning Objectives Neuropeptides – How are neuropeptides made and sorted into regulated secretory vesicles – Know the differences between synaptic vesicles and regulated secretory granules How does slow neurotransmission work – How do G protein coupled recep...

Neuropeptides and Slow Neurotransmission Learning Objectives Neuropeptides – How are neuropeptides made and sorted into regulated secretory vesicles – Know the differences between synaptic vesicles and regulated secretory granules How does slow neurotransmission work – How do G protein coupled receptors work – How do G proteins regulate ion channels – Which ion channels are important for slow neurotransmission – What are the differences between slow and fast neurotransmission. Presynaptic Terminal Post-synaptic Spine Synaptic Vesicles (Neurotransmitters) Active Zone Post-synaptic Density (PSD) G-protein -gated Receptor NeurotransmitterG-protein Gated Channel -gated Receptor Dense Core Vesicle (Neuropeptides) neuropepties can ONLY be released from Dense core vesicles Neuropeptides vs Classical Transmitters While only 9 classical transmitters in vesicles, about 100 neuropeptide precursors (many more peptides) Neuropeptides are made from protein precursors encoded by genes, classical transmitters are synthesized by enzyme in the cytosol Neuropeptides are stored in dense-cored vesicles; Classical transmitters are stored in synaptic vesicles. can evolve a new neuropoptide very easily in comparison to classical transmitters Neuropeptide Precursor Structure Neuropeptide A Signal Sequence every NP starts with a cleavable Signal sequence Cleavage Sites 1-50 Peptides/Precursor you can have many peptides on one precursor, it is actually rare to only have one peptide Neuropeptide B Glycine for Amidation —> this indicates another Post trans. mod. and it prevents degradation when a neuropeptide has to sit for a long time, it i for stability Common Features of Neuropeptides Signal sequence to obtain entry to the ER Precursors with cleavage sites (usually dibasic residues) to release active peptide Use slow neurotransmission (G proteinlinked receptors) Are released from dense-cored vesicles (regulated secretory vesicle) Anatomical distribution of neuropeptides Co-transmitters; most neurons also have classical transmitter. Can be multiple neuropeptides in same neuron. Widely distributed throughout brain -most neurons contain a NP transmitter Neuroendocrine cells in the hypothalamus and pituitary use neuropeptides as primary transmitters (i.e. vasopressin and oxytocin). —> some that don’t have classical transmittes Useful anatomical markers to define subsets of neurons with different properties (i.e. projections, ionic properties, inputs, etc.) – Note GABAergic neuron subtypes are often defined by their neuropeptide co-transmitter (Vasoactive intestinal peptide (VIP), Somatostatin, Cholecystokinin (CCK), neuropeptide Y (NPY): Why are neuropeptides made as precursors? Small peptides cannot be inserted into the ER cotranslationally (minimum of 50 aa to make signal sequence appear out of ribosome) Gives flexibility (multiple peptides/precursor, regulation of processing, mechanism for generating diversity) Evolutionary history (yeast mating peptides have similar structure and processing). Peptide-based communication predates the nervous system. Biosynthesis of Neuropeptides Differences in synthesis lead to storage of peptides in different vesicles than classical transmitters Encoded by genes, transcribed into mRNA, translated into proteins Co-translationally inserted into rough endoplasmic reticulum (ER) followed by signal sequence cleavage Transported through the golgi and then sorted into transport vesicles. Cleavage occurs in the Trans-Golgi network, immature or mature secretory vesicles depending on the precursor. there are NO synaptic vesicles that bud off the TGN —> they are incorporated through endocytosis Secretory Pathway ER Ribosomes Golgi Trans-golgi network (TGN) Clathrin-coated Vesicles; endosome and lysosome Constitutive Secretory Vesicles Translocation, signal sequence cleavage Folding Initial glycosylation Regulated Secretoy Vesicles Glycosylation Sorting, Endoproteolytic Cleavage; Sulfation More biosynthesis of neuropeptides TGN is a sorting organelle, important step for neuropeptides is sorting into regulated secretory vesicles Many vesicles bud from TGN – – – – – – – Regulated secretory vesicles Axon-targeted vesicles Somato-dendritic targeted vesicles Endosmal targeted vesicles Lysosomal targeted vesicles Retrograde vesicles to Cis-Golgi and ER (Note no synaptic vesicles; see next lecture) How are neuropeptides sorted into regulated secretory vesicles? these prohormones (peptides) become to aggregate bc the TGN is more acidic which is a signal for sorting Condensation in TGN (dense cores) segregates neuropeptides from other constituents. May stimulate binding of aggregation-specific proteins that lead to effective sorting. – Unanswered question concerning what regulates their budding (coats? Curvature-inducing proteins?) Followed by budding of other vesicles from immature secretory granules. This does not explain how transmembrane proteins are sorted into regulated secretory vesicles. —> next slide How do TM proteins get into dense-cored vesicles the original vesciles that bud off don’t contain these TM proteins bc they arent sorted by the aggregation There are TM proteins sorted into DCVs as well (some DCVs contain VMAT and store biogenic amines!). It turns out that these are NOT sorted into DCVs at TGN, but probably enter through a specialized endosome  DCV sorting pathway… Details are still being worked out. Neuropeptides are cleaved by specialized proteases you want the prohormone to aggregate and then sort because this is more efficient Furin and Prohormone convertases (PCs) cleave the peptides from their precursos. Furin cleaves in the TGN; PCs cleave in immature secretory granules. PCs recognized a dibasic site and Furin requires an additional basic amino acid Basic X Basic Basic. (Basic amino acids are lysine (K) and arginine (R)). Processing happens throughout the secretory pathway Neuropeptide Signal sequence cleavage (ER) Neuropeptide Furin (TGN) PCs (secretory vesicle) Endoproteolytic cleavage (TGN or secretory vesicle) Neuropeptide Carboxypeptidase (secretory vesicle) —>removes the last dibasic residue Neuropeptide Peptidyl-glycine-a-amidation (secretory —> converts glyceine into vesicle) amine group Neuropeptide NH2 (amide) Regulation of Neuropeptides Regulation at the level of gene expression is predominant; cells can express new neuropeptides through just increasing gene expression. Translational control of mRNAs has been demonstrated for many neuropeptides Release is regulated through many pathways, similar to synaptic vesicles (next lecture). Question of the Day How does increasing expression of neuropeptide affect the amount of neuropeptide released? in general, there should be a limiting size to the vesicles, so there should be an increase in the amount of veiscles and not an increase in the size one you can remake them locally, one you can’t one you can run out of, one you can’t Major difference from classical transmitters is refilling and reuse Packaging at the TGN through sorting does not allow for efficient formation of dense-cored vesicles at synapses – Time for new DCV to arrive at synapse (hours). Cannot release at high frequency; need large store of DCVs; Time course of regulation is slow. In contrast, transporter uptake of transmitter from cytoplasm allows for refilling of synaptic vesicles endocytosed from plasma membrane – Time to re-form Synaptic vesicle at synapse (secondsminutes Can release at high frequency; time course of regulation is fast. What do neuropeptides do? Modulatory, like biogenic amines. Often a feedback control since only released at high firing rates, inhibits presynaptic cell Homeostasis, stress, and reproduction May be important in genetic differences in behavior (e.g. Foraging in C. elegans is determined by polymorphism in they modulate things, and specialize things bc theyre often not neuropeptide receptor) -necessary Special roles in specific behaviors mood, regulating eating, water balance, etc. are regulated by neuropeptides – Opioids --> Pain – Ghrelin, GLP-1, NPY --> Feeding – Orexins --> Sleep Difference between endocrine peptides and neuropeptides Many non-neuronal peptides are processed similar to neuropeptides. – Insulin (Recptor tyrosine kinase, not GPCR). – GLP-1 (made in both neuronal and non-neuronal cells) Regulated release is somewhat different in some endocrine cells although calcium is still important, cAMP is also important Peptide transmitters are ancient and were around before neurons Neuropeptides (and other modulatory transmitters like biogenic amines) use slow synaptic Transmission used by some classical transmitters too G protein-linked receptors mediate slow synaptic transmission (fast transmission uses ligand-gated ion channels) G protein-linked receptors alter the potential of the cell through regulation of ion channels. G protein-linked receptors also modulate many other cellular properties such as gene expression, cytoskeletal dynamics, transmitter release and vesicular trafficking. slow neurotransmission: 1. regulating ion channels 2. directly regulate the mechanisms of fast neuortransmission to alter propability of release Ligands G protein-linked receptors Seven transmembrane receptors Serpentine receptors almost gone Metabotropic receptors glutamate they are for cellular communication Phosphorylation G protein binding and Specificity for downstream effects Internalization G proteins GPCRs are just big GEFs —> they regulate the exchange of GDP to activate G proteins GTPase GDP GTP Hydrolysis Inactive Protein Guanine Exchange Factor (GEF) Activate to GTP GDP GTP Active Protein RGS protein or effectors stimulate inactivation of G protein GTP G protein-linked receptor (GEF activity stimulated by ligand binding) “Large” G proteins there are 3 large subunits, beta/gamma subunit are tight togther very tight a b Ligand g When converted to GTP active state, the alpha will leav,e to become the GTP, bc it is freee to move a GTP b b gg G proteins Three subunits; a, b and g (b and g are always linked) Ga is the G protein, binds to GTP Both Ga and Gbg are effectors; GTP bound Ga is not only an effector, but releases Gbg to be an effector as well Many different Gas, specificity for G proteins and effectors, but three major types. – Gs  increase adenylate cyclase; make cAMP Gs stands for stimulatory – Gi  decrease adenylate cyclase, act directly on ion channels Gi - inhibits – Gq  increase phospholipase C Also many different Gbgs; specificities for Gas and effectors are still not clear. These were the first GTPases to be discovered; that is why Ras/Rac, etc are called small GTPases. G protein Effectors Adenylate Cyclase (makes cAMP from ATP); – regulation of cAMP dependent protein kinase (PKA) – direct regulation of ion channels – regulation of other enzymes (I.e. cAMP-dependent GEFs) Phospholipase C (makes Inositol triiphosphate (IP3) and diacylglycerol (DAG) from Phosphoinositol 4-5 biphosphate (PIP2) – IP3 releases calcium from internal stores (ER) – DAG binds to C1 domains C1 domain proteins (GEFs, synaptic release proteins, protein kinase C (sometimes also requires calcium) Increases release by binding to UNC 13 (See last lecture) Activates other proteins (DAG-dependent GEFS) inhibits cHMP phospho.. Transducin  Cyclic GMP Phosphodiesterase (Specifically for Vision) -transducin is important cuz it does vision Ion channels Second and Third Messengers Ion channels (or any other downstream target) can be modified 3 ways by slow neurotransmission. – Ga or Gbg binding directly to the channel – Direct binding of second messenger to the channel (e.g. cAMP-gated channels) – Phosphorylation of the channel by second-messenger gated kinase (e.g G protein--> phospholipase C --> Diacylglycerol --> PKC --> phosphorylation of channel. Specificity of G protein signaling The effects of ligand binding to any particular G proteinlinked receptors is determined by the specific sub-type of G proteins it binds and the specific effectors of those G proteins. For example, there are two types of norepenephrine/noradrenaline receptors; beta-adrenergic receptors bind to Gs and activates adenylate cyclase, while alpha-adrenergic receptors bind to Gi and inhibit adenylate cyclase. Thus one transmitter, noradrenaline, can activate multiple receptors having opposite effects. Similarly, in the striatum there are neurons express either D1 (dopamine receptor 1) which is Gs coupled or D2 which is Gi coupled. What does slow neurotransmission look like Slow EPSPs Slow IPSPs Shape of Action Potential Size of Fast EPSPs and IPSPs Voltage (post-synaptic) Slow EPSPs Time (seconds) Ligand What channels are effected? Closing Resting K+ channels or Opening Resting Cation channels (Na and K+) Voltage (Post-synaptic) Slow IPSPs Time (seconds) Ligand What channels are most likely effected? Opening resting K+ Channels Voltage (Pre-synaptic) Shape of Action Potential Time (milliseconds) Ligand What channels are effected? Closing Voltage Gated K channels or opening Voltage gated Ca channels Voltage (post-synaptc) Voltage(pre-synaptic) Size of fast PSPs Time (seconds) Time (milliseconds) Time (milliseconds) Ligand Time (seconds) Time (milliseconds) Time (milliseconds) What proteins are effected? Ligand-Gated Ion Channels; Changes in transmitter release What are the G protein Ligands? There are more different G protein linked receptors than any other type of protein Classical Neurotransmitters Neuropeptides Sensory Inputs (Odorants, Taste, and Light) Lipids, e.g. prostoglandins; endocannabinoids (other kinds of neurotransmitters) Cytokines (allows neuro-immune interactions) How long does the signal last? Ligand-gated Channels are regulated by the Inactivation of the Channel (in the ms range). G protein linked receptors can send signals that last from seconds to hours; Multiple levels to regulate time – – – – Desensitization of Receptor (Phosphorylation/Internalization) GTPases to turn off G protein Enzymes to Degrade Second Messengers Phosphatases to reverse actions of kinases Voltage(post-synaptic) Change inChange Resting Membrane Potential in Slow Epsp Time (seconds) Ligand Time (seconds) Ligand Complexity of Control Each neuron may have 20-30 different G protein-linked receptors Allows for incredible fine tuning of neuronal responses by many different modulators Learning Objectives Neuropeptides – How are neuropeptides made and sorted into regulated secretory vesicles – Know the differences between synaptic vesicles and regulated secretory granules How does slow neurotransmission work – How do G protein coupled receptors work – How do G proteins regulate ion channels – Which ion channels are important for slow neurotransmission – What are the differences between slow and fast neurotransmission.