Synaptic Exam 3

what must be true IF (+)ACh was released through channels in cell membrane

• changing DF on ACh by changing ACh concentration gradient should change quantal size (vesicle release)

• changing DF on ACh by changing Vm should change quantal release

• ACh release should cause a change in membrane current (as crosses the membrane thru channel)

evidence against transmitter release through presynaptic channels

• when change in DF on ACh, mEPP amplitude stayed the same. Means no release through channels- and no change in quantal size/vesicle release

• no membrane current is recorded

imaging evidence for vesicular release (stimulate presyn neuron, freeze tissue, electron micrograph image)

formation of omega profiles following motor neuron stimulation

• no stimulation: normal vesicles

• axon stimulation: omega profiles- depicts vesicles fusing with presynaptic membrane

  • shows NT release is quantal- each vesicle fusing out into cleft

from NMJ active zone shows

proteins embedded in presynaptic membrane- stereotypes through frog NMF active zone

• for ex, N-type Ca2+ channels

freeze fracture experiments

imaging evidence for vesicular release- separates the 2 leaflets of lipid bilayer to look inside

• process: chunk of synapse embedded in ice block. cut preparation, which will break at a weak spot. inside, bilayer is structurally weak. two leaflets will be split in 2. look inside lipid bilayer membrane

  • neuron at rest:

    • dots are the proteins that line the active zone (membrane has been cut)

  • neuron stimulated:

    • freeze right after stimulation shows indentation. vesicles in process of fusing with membrane (aka “pits”)

  • neuron 50ms post-stimulus:

    • no more pits because vesicles completely fused with membranes. rows of proteins back in order

how charges accumulate on a cell membrane

• capacitor: structure that can separate charged particles (holds stores/charge)

• capacitance measured in farads

• capacitance of cell membrane is directly proportional to surface area of the cell (approx 1 uF/cm2)

  • larger cell, increased membrane, increased capacitance

capacitance increase with increased surface area

as synaptic vesicle fuses with membrane for release, surface area increases (as vesicle membrane becomes part of cell membrane) and increases capacitance

amperometry

measure of current

• comes from release of NT when norepinephrine is released and undergoes a redox reaction

  • redox rxn produces carbon fiber, which can measure current and capacitance

capacitance as a measure of vesicle fusion

large chromaffin granules- large to increase cell size and increase capacitance for measurement

• current is produced upon release of norepinephrine and carbon fiber reads it

• between norE releases, capacitance jumps (increases)

release of transmitter causes

• increase in capacitance

• increase in current

  • evidence for vesicular release

capacitance as a measure of vesicle fusion

• stimulate presynaptic neuron

• causes simultaneous inward current of post synaptic neuron

  • presynaptic neuron increases capacitance (+ surface area) and at same time postsynaptic neuron produces EPSP

(same for mini EPPs- stimulate presynaptic neuron causes excitatory postsynaptic response)

by averaging several events of capacitance changes, a small change can be detected at nerve terminal following nerve stimulation

• will find presynaptic stimulus capacitance increases- matches EPSP of mini

  • shows vesicular release, even for single release (mEPP)

overview process of synaptic vesicle fusion and exocytosis

synaptic vesicles —> docking —> priming —> calcium triggered fusion —> exocytosis

SNARE proteins that form CORE complex

3 SNARE proteins:

• syntaxin, SNAP25, synaptobrevin/VAMP

  • syntaxin and SNAP25: associated with (on) plasma membrane

  • synaptobrevin/VMAP: vesicle associated (on) membrane protein

they dock the vesicle on the intracellular surface of the plasma membrane by connecting the alpha helices coils of each into a coiled core complex

vesicle docking- Munc-18 and SynPrInt site

• several chaperone proteins bring synaptic vesicles to active zone

• Munc-18 keeps syntaxin in folded configuration until vesicle ready to dock

• SynPrInt site on Ca2+ channel binds to folded syntaxin and other CORE proteins, which allows Ca2+ channels to be closely associated with docked vesicles

normal synprint binding vs synprint peptides binding

normal: synprint site of voltage gated calcium channel binds folded syntaxin

synthesized SynPrInt peptide: matches the part of syntaxin that binds to calcium channel, so binds there and prevents syntaxin from binding

• reduces release and abolishes synchronous release

vesicle priming

step in between vesicle docking and vesicles being ready for release

• synaptic vesicle is docked to membrane and synaptotagmin (calcium sensor binding domain on vesicle- where calcium binds to vesicle) C2A and C2B bind to CORE complex (coiled core complex alpha helices)

  • complexin also binds at this step (forms complex between synaptotagmin and coiled complex)

calcium entry and vesicle fusion

• calcium enters the cell and ions bind to synaptotagmin (C2A and C2B sites)

• triggers vesicle fusion and release of transmitter into synapse

synaptotagmin, syntaxin, SNAP25, VAMP purpose

bring vesicle down to fuse with presynaptic membrane

synaptotagmin as calcium sensor for vesicle release

• wild type synaptotagmin:

  • downwards peak = fast synchronous transmitter release (current very smooth and transmitter release at same time)

  • smaller peaks = asynchronous transmitter release

• synaptotagmin-1 knockout

  • results in ONLY asynchronous release- tells us synaptotagmin-1 is necessary for synchronous vesicle release

C2B for vesicle release

• wild type C2B

  • slope of 3.5 between number vesicles released and calcium concentration (about 4th order)

• C2B mutation (decrease affinity to Ca2+)

  • still binds calcium but at decreased affinity- takes more calcium to cause same amount of vesicle release (slope 3.6)

• C2B removal

  • calcium binding domain removed entirely, slope 1.6 means C2B crucial for 4th order relationship.

endocytosis and why

process of membrane being brought back into the nerve terminal

• to regulate the increase in surface area during exocytosis

protein interactions for reverse of vesicle release

• alphaSNAP: form around CORE complex

  • coats coiled complex and identifies bonds to be broken

• NSF: ATPase untangles CORE proteins

  • ATPase hydrolyzes ATP for energy to undo coiled core complex

evidence that endocytosis and exocytosis occurs at same time

• stimulate for 10Hz for 1 min

  • moderate level stimulation, lots of vesicle release, extent of active zone expanded, presence of endosomes (large vesicle of retrieved membrane) tells us endocytosis occured

• stimulate for 10Hz for 15 min

  • almost out of vesicles, extant of active zone inc, takes on funky shape because cant retrieve membrane that quick, many more endosomes in terminal (proves endo and exo at same time)

which part of membrane needs to be retrieved

doesnt matter as long as you maintain size and shape

pH effect to check endocytosois

synaptopHluorin fluoresces as pH increases (less acidic)

• it fluoresces at 7.2 (outside cell) but not at 5.5 (inside cell), so when in extracellular fluid, it fluuresces.

• when it comes back into the cell (cell acidifies) so stops fluorescing

spH in mouse NMJ

• few seconds after stimulation (15-30) bright fluorescence around axon terminals bc vesicle fusion

• starts to fade at 60-75 seconds, bc the membrane that fused has now endcytosed and re-acidified, so fluoresncence goes away.

  • membrane being retreieved is SAME vesicle that was released

freeze fracture image significance and what it shows

coated pits- membrane in the process of being endocytosed.

• coated in protein, to say that is the membrane that needs to be retrieved.

coated pits are NOT in the active zone- endocytosis occurs away from active zone where exocytosis occurs (cant do at same place)

3 methods for recycling vesicle membranes

• kiss-and-run (at synapses that arent very active)

• clathrin-mediated (most common

  • these 2 wont occur at same synapse

• bulk (occurs, but not clear physiologically)

clathrin-mediated endocytoses

• AP2 (adapter protein 2) interacts with Ca2+ bound synaptotagmin

  • perfect marker for what type of membrane to bring back (bc that vesicle was j fused out)

• clathrin binds to AP2 to endocytose membrane

  • PIP2: essential for clathrin to work properly (exact mech unclear)

• clathrin molecules join together into triskelion complex and form spherical complex

  • pulls part of membrane to bring back into the cell

clathrin mediated endocytoses coating of pits

• AP2 binds to synaptotagmin and tugs on membrane

• clathrin binds to AP2 and tugs in membrane to bring it in

• dynamin pinches end of membrane to form a vesicle —> coated vesicle

• uncoating as ca2+ undbinds to synatoptagmin and dissociates

vesicle pinching by dynamin

dynamin molecules form cluster where pinching off will occur: 2 paths

• GTP hydrolysis: allows separation to actually happen

• GTP-gammaS: allows GTP to do its activity, but prevents hydrolysis so separation never occurs and dynmin accumulates into “tower”

bulk endocytosis transmitter release

can occur if massive amounts of vesicle release

• syndapin proteins bind to membrane and pull in a big piece of membrane —> forms endosome

• endosome breaks off into pieces forming synaptic vesicles (in active zone)

kiss-and-run transmitter release hypothesis

occurs at less active synapses

• docking complexes are holding vesicle to membane, the vesicle starts to fuse “kisses” the membrane, releases some NT, then goes back into terminal

  • doesnt release all its NTs

3 parts of vesicle pools

• ready pool

  • docked vesicles with coiled core complex holding it to membrane. ready for release (after priming and Ca2+)

• recycling pool

  • vesicles that have been endocytosed and are empty. need to be filled with NT, have no CORE proteins, but once added can be part of ready pool

    • no synapsin, so not tethered to cytoskeleton

• reserve pool

  • vesicles in reserve (backuops) that aren’t ready to be release but also weren’t just recycled

    • are tethered to cytoskeleton bc synapsin

synapsin phosphorylation

synapsin dephosoprylation

• caused by decreased calcium, increases tethering, more vesicles in reserve pool than recycle

synapsin phosphorylation

• caused by increased calcium, decreases tethering, more vesicles in recycle pool than reserve

botulinum and tetanus toxin targets

cleaves proteins of core coiled complex (synaptobrevin/vamp), prevents complex from forming, prevents docking and transmitter release

selective entry of botulinum toxins into nerve terminal

during endocytosis, there are both heavy and light chains

• heavy chain internalizes into the cell'

• light chain gets into cytoplasm and cleaves SNARE proteins

paralyzes, so no more release of NT/movement

ionotropic channel

ligand-gated channel

ionotropic channel characteristics

• rapid onset of effects (channel opening is fast reception)

• rapid termination of effects

• 1:1 relationship between action and response (1 binding : 1 action)

• effects limited by type of ion channel (only cl- ions pass through cl- channel)

• binds larger (uM) concentration bc located near site of NT release (in active zone)

  • —> lower affinity

metabotropic

g-protein-coupled receptor

• not an ion channel

metabotropic receptors

• slow onset of effects (bc series of processes)

• slow termination (all processes need to inactivate)

• greater than 1:1 response

• multiple diverse effects from single NT due to multitude of second messenger signaling pathways

• usually bind NT in nM range (smaller concentration) bc located far from site of NT release

  • higher affinity

families of ionotropic receptors has to do with

structure

families:

• pentameric (cys-loop) receptor

• glutamate receptor

• trimeric receptor

• TRP receptor

pentameric (cys-loop) receptor

• pentamer, 5 subunits, 4 transmem segments

  • nAChRs, seratonin, GABAa, glycine, ZAC

glutamate receptor

• tetramer, 4 subunits, 4 (including one small) transmembrane seg

  • NMDA, AMPA, kainate

trimeric receptor

• trimer, 3 subunits, 2 transmem seg

  • ATP, ASIC

TRP receptor

• tetramer (but diff fam from glutamate), 4 subunits, 6 transmem, 1 p-loop

  • TRP receptors

ACh receptor basics

binds ACh extracellularly, stingrays are good source of ACh

nAChR subunits

gray circile subunit (5), with 4 transmembrane seg, M1-4 segments

M4 segment closest to core and pore lining so determines ion selectivity

• pentameric family (AChR)

ACh binds to alpha subunits + receptor needs 2 ACh to bind, so each ACh receptor has at least 2 alpha subunits

combinations of neuronal nAChR subunits

homomeric

• 5 identical subunits (alpha 7 or alpha 9)

heteromeric

• the 5 subunits may be diff, as long as each has 2 alpha subunits

ligand binding

• probabilistic, so binding frequency increases with concentration of ligand

• amount of time ligand spends bound to receptor varies by ligand (KD)

what binds to ACh receptors

• ACh binds all ACh receptors

• nicotine binds only ionotropic ACh receptors aka nAChR

dissociation constant KD

• strength of binding affinity

• Koff / Kon

• concentration at which 50% binding sites are occupied

• higher KD means lower affinity

using graph to find KD and affinity

• find affinity for nAChRs in neurons vs muscle

look at 50% receptors occupied on y-axis

• KD for nAChRs in neurons req lower concentration ACh —> higher affinity

• KD for nAChRs in muscle req higher concentratuon ACh —> lower affinity

ligand agonists

compound that elicits same biological effects as the endogenous (naturally occuring) ligand when it binds to receptor

• nicotine or ACh: Vm will be same when agonist nictone is used bc has same effect as when ACh used

ligand antagonists

compound that reduces or eliminates effect of an agonist when bound to receptor

• tubocurarine or alpha bungarotoxin

  • reduces of abolishes Vm response

competitive antagonist

binds to same site as agonist (orthosteric binding) but does not activate the receptor

reduces or prevents activation of receptor by an agonist

non-competitive antagonist

binds to the receptor at a different site from an agonist (allosteric) but prevents or reduces activation of the receptor

NAM: negative allosteric modulatpr

reversible antagonist

binds non-covalently to the receptor, so can come off by “washing off”

• tubocurarine

irreversible antagonist

binds covalently to the receptor, so cannot be displayed by either competing ligands of “washing off”

• alpha bungarotoxin

GABA receptors

• inhibitory but still in pentameric family

• GABA B: metabotropic

• GABA A: ionotropic and pentameric family

  • are ligand gated chloride channels

• equilibrium potential of GABA receptor is same as that of chloride

effects of GABA A receptors: hyperpolarizing inhibition

• DF on GABA receptors determined by E_Cl

• DF gaba = Vm = Ecl

  • chloride current (~ -70mV) will always move membrane potential towards its equiibrium

• VM -40 to -50 is hyperpolarizing (bc cl- coming into the cell)

  • VM -80 is depolarizing, but is still inhibitory because can only depolarize to at most -70, which is hyperpolarized compared to AP threshold

effects of GABA A receptors: shunting inhibition

• gaba receptors not activated:

  • normal AChR release (excitatory, depolarizing)

• gaba receptors activated:

  • AChR will cause decreased excitatory response because of decreased membrane resistance/ increased conductance which reduces voltage (V=IR)

gaba receptor effect when Vm = Ecl

inhibitory, even when no net current

inhibition with ion channels

IPSPs generation when ion channels are opened causing hyperpolarization of membrane ??

open gaba receptors reduces excitatory effects —> inhibitory effect

• ACh released at distal dendrite

  • EPSP, decays with distance towards soma, depolarizes cell more tha Ecl, so Cl enters the cell to bring back to -70

    • glutamate also tries to depolarize, but has less of an effect with GABA receptor being open

• ACh released close to soma

  • inhibitory synapse closer to soma, so inwards movement will decrease response by more?

glycine receptors (GlyRs)

inhibitory ionotropic receptor in pentameric family

• glycine vs GABA current

  • glycine is faster to activate and desensitize?

ionotropic glutamate receptor structure

• tetramers- 4 subunits, has large extracellular regions

  • includes NMDA and AMPA

• 2 extracellular domains

  • first: ligand binding domain where cofactors bind

  • farthest: amino terminal domain- site of modulation for glutamate receptors’ functioning

NMDA vs Non NMDA ionotropic glutamate receptors

Non-NMDA (AMPA and kainate)

• low conductance, fast gating and densitization, permeable to Na+/Ka+ and sometimes Ca2+, not subject to Mg block

  • faster neurotransmission due to AMPA receptors

NMDA

• high conductance, slow gating speed and desensitization, permeable to Na+/K+/Ca2+, subject to Mg block

  • requires both glycine (plentiful) and glutamate to bind to activate

  • excitatory post synaptic response inwards current due to Ca2+ influx

Mg2+ block of NMDA glutamate receptors

• at resting potential, NMDA receptors are blocked by Mg2+

• Mg2+ gets pushed out when cell gets depolarized (excitatory input)

• second glutamate binds to NMDA receptor which opens channel and passes current

NMDA receptors essential for

Ca2+ influx

NMDA receptor only passes current when

presynaptically: glutamate release

postsynaptically: depolarization

IV plot for NMDA receptor

linear without Mg (similar to AChR iv plot)

with Mg: little to no current until enough depolarization to remove Mg block