Neurotrasnmitter Release and Recycling

H+ proton Gradient for loading vesicles with NTs

• NTs get pumped into secretory vesicles by antiporters
• think just like the ETC = Electron transport chain the secondary active transport is given by Hydrogen protons H+
  --> H+ protons are pumped into the vesicles by ATP pumps
• Co-transporter then anti-ports H+ ions out and NTs in
  
  

Vesicle General cycle

• vesicles dock at active zones
  --> active zones are areas where voltage-gated calcium channels are dense so the vesicles can be reached easily by calcium for release
• are primed for release
• then fuse withe the membrane
• then they bud off the membrane, fuse into endosomes, and again bud off endosomes to resume cycle
  
  

Distinct Pools of Vesicles

• Reserve Pool of vesicles
  --> a reserve pool of vesicles are held away from the active zones by actin filaments
  --> readily releasable pool are at the active zones and begin docking through the RAB- RIM proteins
  
  
  

SNARE Proteins, Docking and Release

• RAB- RIM proteins
  --> initial docking starts when the Rab protein on vesicle binds to RIM protein on terminal membrane
  --> think the vesicle wants to gRAB = RAB the RIM of the membrane
• V-SNARE protein = vesicle protein
  --> synaptobrevin
• t-SNARE proteins = target proteins
  --> syntaxin
  --> SNAPP-25
• SNARE Complex
  --> the SNARE complex is formed by the v-SNARE and t-SNARE proteins binding to form a helix
• Release TAG protein
  --> synaptotagmin
  --> note that synaptotagmin without bound calcium is slightly attracted to the t-SNARE proteins
  --> Calcium binding to synaptotagmin increases the affinity of synaptotagmin for the t-SNARE proteins
  --> Calcium enters through voltage gated calcium channel at the active zone
  --> Ca2+ binds to synaptotagmin and the vesicle fuses with the plasma membrane
  
  

Recycling of the Vesicle

• NSF is special protein that disassembles the SNARE complex so the vesicle can be recycled
  --> think of NSF = "NO SNARE FUSION"
  
  

Botulism and Tetanus

• both botulism and tetanus stop vesicle fusion and thus NT release
  --> they selectively cleave SNARE proteins to stop vesicle docking
• both come from clostridium bacteria that release toxins
  --> clostridium botulinum
  --> clostridium tetani
• death comes from no NT release for muscles
  --> mainly die from respiratory/diaphragmatic failure
• Botulism
  --> cleaves SNARE proteins in motor neurons
  --> facial BOTOX is Botox-type A
• Tetanus
  --> cleaves SNARE proteins in inhibitory spinal cord neurons (Glycine mainly)
  --> causes sustained contraction
  

NOTES
Ionotropic and Metabotropic NT Receptors

IntroductionfdIonotropic receptors = ion channel receptors
--> directly allow ions to pass through

• Metabotropic receptors = GPCR = G protein coupled receptors
  --> have cascade signals that amplify
  --> think GPCRs have long term effects and will effect metabolism = metabotropic
  
  

Classifying Ionotropic and Metabotropic Receptors

• the main excitatory and Inhibitory NTs and their main receptors are ionotropic with the lesser known ones being metabotropic
  --> think that they are the main NTs for generating or stopping APs, thus they act directly through ions
• GABA receptors
  --> GABA-a is ionotropic and allows in Cl- ions
  --> GABA-b is metabotropic
• Glycine receptors are ionotropic
  --> being the other inhibitory NT, Glycine receptors are similar to GABA-a in that they are also ionotropic and also let in Cl- ions to hyperpolarize

• Glutamate receptors

  
  

  --> main Glutamate receptors: NMDA, AMPA, and Kainate are all ionotropic

  
  

  --> lesser known mGluR is metabotropic

  
  

• Acetylcholine receptors are based off their names

• nicotinic ACh receptors
  --> start with an N so they are Ionotropic

• muscarinic ACh receptors

  
  

  --> start with an M so they are metabotropic

  
  

• Monoamine NTs and All Neuropeptides (opioid agonists)

  
  

  --> ALL are metabotropic = GPCR

  
  

  --> again think Monoamines start with an M

  
  

  --> dopamine, serotonin = 5HT, NE

• the only exception is serotonin subtype 3 is ionotropic
  
  

Metabotropic = GPCR Receptors

• have 2 actions
  --> modulate other ion channels
  --> start cascade effects for protein synthesis, etc.
• Trimeric GTP binding proteins = alpha, beta, and gamma
• Alpha subunit
  --> has a bound GDP to it
  --> once NT binds to GPCR, Alpha unit replaces GDP with GTP
  --> when GTP is bound to alpha, it disassociates from beta and gamma
  --> alpha causes downstream effects and cascade
  --> ex: Adenylate Cyclase
• bound beta and gamma subunits modulate other membrane proteins or ion channels
  
  

2 Most Common GPCR Pathways

• cyclic nucleotide pathways
  --> cAMP or cGMP
• phosphoinositol pathway = Phospholipase C Pathway
• note it is the alpha subunit type that determines what "primary effector" a GPCR will effect
• Main GPCR types:
  --> Gs = cAMP stimulation through Adenylate Cyclase
  --> Gi = cAMP inhibition through inhibiting Adenylate Cyclase
  --> Gq = Phospholipase C pathway
• note 2ndary effectors are always kinases
  
  

Gs cAMP GPCR Pathway

• primary effector = Adenylate Cyclase
• 2nd messenger = cAMP
• secondary effector = PKA = protein kinase A
• final effect = increase protein phosphorylation
  --> since the 2nd or final effector is a kinase
  
  

Gq cAMP GPCR Pathway

• primary effector = Phospholipase C
  --> note PhosphoLipase C stands for CHOPPER since this protein chops the head off of PIP2 = a lipid that makes DAG and IP3
• 2nd messenger = Diacylglycerol (DAG) and inositol triphosphate (IP3)
• secondary effector = PKC = protein kinase C and Ca2+ release
• final effect = increase protein phosphorylation due to kinase and also increase Ca2+ levels
  --> note the C in Phospholipase C is for both the kinase and for the Calcium release
  
  

4 Ways Ca2+ levels are controlled and kept low in cell

• Ca2+ ATPase pumps calcium out of the cell
• Na+/Ca2+ exchanger at the cell membrane uses the sodium gradient to pump calcium out
• calbindin is a bufferring protein that bufffers Ca2+ in the cell
• Endoplasmic Reticulum stores intracellular calcium
  
  

3 effects of 2nd effectors (protein kinases) on ion channels

• Direct modualtion of ion channels by kinases
  --> phosphorylating them modulates them
• Indirect modulation of ion channels by kinases
  --> kinases may phosphorylate another protein that woul increase or decrease ion channel expression
• Transcriptional regulators of ion channels
  --> may increase or decrease the production ion channels
• examples:
  --> Beta NE GPCR Gs type --> PKA directly phosphorylates K+ channels
  --> stops K+ from leaking out of the cell

Ionotropic and Metabotropic NT Receptors (cotinued)

--> this lowers the threshold for APs in the heart to allow it to pump faster or harder
--> possible for weaker EPSPs = excitatory post synaptic potentials to give an action potential

• GABA-b GPCR receptors - 2 effects
  beta gamma subunits directly activate K+ channels or deactivate Ca+ channels for inhibition
• PKA downstream casues dephosphorylation of NMDA glutamate receptors to stop allowing them to bring in Ca2+
  --> recall the NM in NMDA stands for NEED MORE OR NEED MEMORY (more cations other than Na+ and K+ let in by AMPA glutamate receptors)
  
  

CREB protein

• CREB = cAMP response element binding protein
• transcription factor that becomes activated when phosphorylated by PKA
• CREB is a key protein in memory formation for long term potentiation
  
  

Main GPCRs types

• recall Glutamate, GABA-a, Glutamate, 5HT type3,nicotinic are ionotropic receptors
• All Monoamine (minus type 3 5HT) and neuropeptides are GPCRs
• Dopamine
  --> D1,5 = excitatory = Gs
  --> D2,3,4 = inhibitory = Gi
• NE
  --> Beta 1,2 = Gs
  --> Alpha 1 = Gq
  --> Alpha 2 = Gi
• ACh Muscarinic
  --> M1,3,5 = MNOPQ = Gq
  --> M2,4 = Gi