ligand-gated ion channels and fluorescence

what are neurotransmitters

chemical messengers released from one neuron (‘presynaptic cell’) and acting at a close site on another (‘postsynaptic cell’) to elicit an effect determined by the specific nature of the receptor

-a local and specific action

types of NTs

Mostly aas, peptides and monoamines:

- Acetylcholine: voluntary movement of the muscles

- Glutamate: major excitatory NT, roles in memory and learning

- Dopamine: motivation, pleasure associated with addiction and love

- Serotonin: emotions, wakefulness and temperature regulation

- GABA: the major inhibitory NT

- ATP: neuronal/glial communication, role in pain regulation

-termed agonists: ligands that cause a postsynaptic effect

-referred to as endogenous agonists as they are made by the body

-antagonists are ligands which block the action of the agonist

ionotropic vs metabotropic

-NTs are ionotropic receptors

-ionotropic have the ligand bind on the structure which is an ion channel

-metabotropic receptors are also membrane bound but binding triggers a cascade binding effect

quantal release

-the smallest postsynaptic response arises from the release of NT from one vesicle

-the contents of one vesicle of NT is referred to as ‘quanta’

-single vesicles are released spontaneously and their postsynaptic response is called a ‘mini’

-what dictates the decay of the currents?

- Persistent Ach application produces persistent current; not indefinitely though, the receptors to eventually desensitise

- Diffusion dominated processes have a Q10 of 1 (ratio between the timing of a process at room temperature vs body temperature)

- Anything enzyme or protein related often has a very different temperature sensitivity; in this case the Q10 was 2.8

- Also voltage dependence of decay (faster at more negative holding potentials)

- Conclusion was that Ach is actively cleared from the cleft

Ach receptors

Nicotinic Acetylcholine (Ach) receptors

-found at the NMJ and between pre and postsynaptic cells in the ANS

-permeable to Na+, K+ and Ca2+

-composed of 5 subunits – adult form is 2α, 1β, 1δ and 1ε

-α subunit binds Ach (therefore 2Ach bind per receptor)

-antagonists = snake venom and curare compounds, extremely poisonous

myasthenia gravis

-autoimmune disease

-body produces autoantibodies against nicotinic Ach receptors

-EPP can’t generate muscle stimulation

-2^nd subset of MG = autoantibodies against muscle specific kinase (MuSK)

GABA and glycine receptors

-both have pores which allow negative ions passing through

-GABAA, GABAC and glycine are ionotropic, (GABAB is metabotropic)

-since ECl is close to Em and below action potential threshold, they tend to be inhibitory

-pentameric structures

5GT3 receptors

-pentamers

-similar transmembrane loops

-Na+, K+ and Ca2+ permeability

subunit composition dictates…

1. Receptor properties e.g. sensitivity, conductance, kinetics of opening and closing etc

2. Cell surface distribution

3. Dynamic regulation

-δ subunits are sensitive, with low desensitisation; mediates tonic GABAergic currents

pharmacological modulation

-experiment with barbiturates and diazepam both of which dramatically enhance GABAergic response

-recording from a cell and triggering a current

-much larger current with barbiturates

-stuck electrodes to the side of the cell so they can record the opening and closing of the individual ion channels

-you could characterise the opening and closing of the channels, you can measure the length of time they stay open

-did this for thousands of opening and closing events

-the histogram gets splayed out; this shows that each time they open, they tend to stay open for a longer period of time

-the barbiturate action potentiates the GABA receptor by basically locking the receptor open

-however doesn’t alter the duration of the closing time, so it doesn’t ease the opening, but once the channel is open it locks it open

-benzodiazepines eases opening of receptors

purinergic receptors

-metabotropic = P1, P2Y

-ionotropic = P2X

- Agonist is ATP

- Different structure to other ligand gated ion channels

- -Na+, K+, Ca2+ permeability

- Widespread glia and neuronal expression

- ATP released in synaptic vesicles

- An important means to neuronal to glia, and glia-to-glia communication

glutamate receptors

principal excitatory NT in vertebrate nervous systems

ionotropic glutamate receptors

transient opening of ion channels allowing net influx of cations, generating an excitatory current

metabotropic glutamate receptor

modulatory role in synaptic transmission

Inotropic glutamate receptors (iGluRs):

-3 main classes: AMPA, NMDA and kainate receptors

-the classes are agonists, the molecules are exogenous ligands and they are manmade

-glutamate is the molecule used by the body

-glutamate and kainate activate all three types of receptor

-NMDA and AMPA are specific agonists

-KARs are named by exclusion (kainate agonist = no effect of AMPA or NMDA)

-AMPARs and NMDARs are usually co-localised at synapses where they mediate ‘fast’ chemical synaptic transmission

-NMDARs, AMPARs and KainateRs can be both synaptic and extrasynaptic

-GluRs are mainly localised to postsynaptic sites

-the receptors can be on these little blebs where the membrane potential is much more significantly altered in these than overall in the cell

-the receptors are not working in isolation, they are in a large protein network

-they are multimeric protein complexes, comprised of 4 subunits (tetramer)

 3 transmembrane domains (TM1, TM2, TM4)

 A re-entrant loop (TM2)

-loops are formed between the transmembrane sections

-the loops and the extracellular domains are the communication to the outside of the cells, binding sites are formed by the loops

-intracellular are the sites where the receptors are being modulated by the intracellular events

AMPAR subunits

AMPAR subunits: GluA1-GluA4

-tetramers

-each subunit can bind one molecule of glutamate, it binds between the S1 and S2 extracellular domains

-approx. 900aa, 68-73% identity

-can undergo 2 modifications: alternative splicing or RNA editing

-can form homomeric and heteromeric channels

-in heteromeric channels the presence of edited GluA2 determines the I-V (current-voltage) curve and the Ca2+ permeability

alternative splicing

-some exons can be lost during splicing

-splice variants affect the receptor kinetics

-current flow through AMPARs can be terminated by two mechanisms:

1. deactivation: agonist unbinding leads to closure of channel, requires the removal of transmitter

2. desensitisation: the channel closes while agonist remains unbound

-rate of desensitisation is strongly influenced by subunit composition and the flip/flop variant

AMPAR subunit composition affects Ca2+ permeability

-experiment using frog oocytes, injected with RNA for different GluRs

-was recorded in either high Na+ or Ca2+

-GluA2 confers impermeability to Ca2+

Editing at the Q/R site determines the Ca2+ permeability of AMPA receptors

-GluA2 receptors are edited at the Q/R site

-DNA of GluA2 codes for glutamine Q, but this is swapped for arginine R by the editing of mRNA

-the positively charged arginine R renders channels containing GluA2 impermeable to Ca2+

-most AMPARs contain GluA2, but not all

-Ca2+ permeable AMPARs are found on bergman glial cells, some hippocampal neurons, some auditory neurons etc

-implications for plasticity and excitotoxicity

kainate receptors

-less is known about them

-large presynaptic presence

-lower conductance than other GluRs

-may have combined ionotropic and metabotropic actions

-evidence from pharmacological manipulation of 2^nd messenger systems, supported by the absence of effect in knockout animals

NMDA receptors

NMDA receptors = non-specific cation channels, with a large Ca2+ component

-magnesium can also end up being drawn into the receptor and it acts as a wedge that blocks the channel

-this wedge is only released if the cell is depolarised

-similar to the voltage-dependency of sodium channels

-when there is a negative membrane potential, there is no current passing

-as it is further depolarised there is a linear current moving

-zinc and protons modulate the behaviour of NMDA receptors

-voltage-sensitivity turns NMDA-Rs into ‘coincidence-detectors’

long term potentiation

-for the receptors to open, you need activation of the presynaptic cells to allow release of glutamate; at the same time depolarising the postsynaptic cells

-only when both are depolarised at the same time that you remove the Mg cork and allow calcium to enter the cell

-when calcium enters, it carried messages

-this is used in altering the strength of synapsis and the basis in learning and memory

-strong, continuous activation of this pathway then generates APs through AMPA receptors in the post-synaptic cell

-allows calcium to enter through NMDA channels, causes a conformational change and potentiates the pathway; such that after the burst of APs, when you do the test stimulation, the post-synaptic response is much stronger

how is calcium doing everything

-Ca²⁺ is a common ion

-easy to construct proteins which bind Ca²⁺ and change shape

1. Compartmentalisation of Ca2+ entry

2. Localisation of Ca2+ binding proteins

3. Importance of mitochondria

compartmentalisation of ca2+ entry

-neurones particularly, compartmentalise where calcium enters

-only have calcium-binding proteins in certain areas

-summation also happens

-if you had the pre and psot-synaptic activity slightly separated then you got 2 separate small calcium transients

-if they happened at exactly the same time, you got a singular, shorter really large calcium transient

-reason is that there’s 2 different calcium binding proteins directly underneath the synapse, if they happen at the same time, the enormous calcium signal will be detected by calcium binding proteins that are relatively insensitive

-the low level signal is detected by more sensitive calcium binding proteins; the lower sensitivity calcium binding proteins don’t get to bind it therefore causing a separation of the message

importance of mitochondria

-adept at sucking up excess calcium

-act as buffers

-at sites with lots of mitochondria, there can never have a high level of calcium

-pre LTP there are no mitochondria in the compartments, after LTP mitochondria move in

luminescence

emission of light by a substance not resulting from heat e.g. fireflies

bio-luminescence

luciferase – firefly tails, aequorin – jellyfish

chemi-luminescence

glow-sticks

phosphorescence

slow emission of light that has been previously absorbed by a substance e.g. watch hands; light emission after illumination

fluorescence

emission of light by a substance that has absorbed light (fast 0.5 to 20ns); light emission during illumination

auto-fluorescence

fluorescence from naturally occurring molecules in your sample

electrons in fluorescent dye

-the electrons in a fluorescent dye molecule they absorb light, and they get to an excited state

-when they relax back down, they give off photons of light

-fluorescein is a universal fluorescent dye

filter cube in microscope

-white light comes in and hits an excitation filter

-everything bar the blue light gets filtered out and blue light gets reflected off this special mirror, focused by the objective lens onto the specimen, the dyes fluoresce green, the objective collects the green light

-the green light passes through the dichroic mirror and then you can see it

-a dichroic mirror reflects below a specific wavelength and transmits above it

confocal microscope

-only selecting the plane of interest

-use lasers as the excitation source, scan a laser point very quickly around the image

-detect the light coming off

-using a pinhole in front of the detector to filter out anything above or below the focal plane

ways to overcome resolution limit

structured illumination (SIM), stimulated emission depletion (STED), localisation (STORM and PALM)

problems

-getting the probe to the target e.g. if its inside the cell

-only labelling the target not everything

-overcome any sample autofluorescence

-phototoxicity – live-cell consideration

-photobleaching, some dyes are more resistant than others

how to get the dye to the target

dye chemistry

antibodies - immunofluorescence

dye chemistry

-live-cell imaging applications

-get through cell membrane

-only become fluorescent in certain environments

-accumulate in certain organelles

-very easy to use and visualise

e.g. Tubulin – tubulin tracker green; ER – ER tracker red; DNA – Hoechst; plasma membrane – cell mask deep red

problems with dye chemistry

- Limited retention time in the cell/organelle

- Limited targets

- Specificity

- Toxicity

antibodies - immunofluorescence

-traditionally don’t exist with the host

-raising them against the protein that we’re interested in

-first fix the cells using formaldehyde

-use a mild detergent to make the cell permeable to the antibody

-block everything else using an excess of ‘non-specific’ protein

-antibody binds to protein

-can add the fluorescent dye directly to the antibody, but a better way is to raise a secondary antibody that recognises the primary antibody

problems with antibodies

- Small-molecule chemical probes – many cannot be fixed, few specifically target individual proteins

- Immunofluorescence techniques – difficult to use with live cells, only cell surface proteins are visible; getting the label to the target requires ‘permeabilisation’

fluorescent proteins

GFP

-target gene DNA is manipulated to contain the code for GFP

-host cell ‘transiently’ expresses GFP-tagged gene or tagged-gene gets incorporated into genome

-DNA is inserted into the cell – virus, liposomes, electroporation, micro-injection

-used in zebrafish (model organism) to look at developmental biology, cancer, genetics etc

problems fluorescent proteins

- Fusion constructs: not native proteins, strong promoters can ‘enhance’ signal, transient transfections – higher expression, may perturb protein function

- Over-expression artifacts, is the protein found in unexpected areas?

dynamic imaging

e.g. live organelle/ protein tracking

-requires chemical or genetic (e.g. GFP) tag to label organelles or proteins

-requiring many images per second

problems with dynamic imaging

- Requires short exposure times

- Bleaching issues

- Toxicity/phototoxicity

- How do you know that the proteins/organelles are active

colocalisation

-can only claim colocalization down to the resolution of the microscope

-do proteins that share the same space interact?

-colocalisation microscopy only suggests this

-using super resolution of microscopes to look at colocalization

FRET

Fӧrster Resonance Energy Transfer

-visualise molecular association and interaction

-established technique to measure the interaction and location of the interaction of 2 proteins or structures

-typically one structure is labelled with a donor fluorophore the other an acceptor fluorophore

-when the proteins come together you don’t get cyan out, you get yellow out

-very very small

-exciting the electrons to the excited state

-the emission spectra of the donor must be matched with the excitation spectra of the acceptor

-typical FRET pairs:

• Fluorescent proteins: CFP (donor) and YFP (Acceptor)

• Fluorescent dyes: fluorescein (donor) and rhodamine (acceptor)

-when the two structures become associated (<10nm) energy transfer takes place from the donor to the acceptor

FRET to visualise cAMP signalling

-problem is there are no fluorescent cAMP reporter molecules

-tagged YFP to the catalytic subunit of PKA and CFP to the regulatory subunit

-no cAMP = FRET

-when there is cAMP there is no FRET – the cAMP binds this molecule and the catalytic subunit gets released meaning no FRET

-introduced DNS for CFP-R and YFP-C PKA subunits into the cell

Ca2+ dyes

fluo-3

fura 2

GcAMP

fluo-3

-fluo-3 is a derivative of fluorescein

 Single excitation, single emission so its fast imaging

 Only fluoresce when bound to calcium ions

 Large increase in fluorescence when bound to calcium ions

 Can cause photobleaching and is difficult to accurately measure calcium ion concentration

fura 2

-Fura 2 is excited at 340nm and at 380nm, when it binds to calcium these properties change

 Dual excitation for a single emission

 Only fluoresces when bound to calcium ions

 Large increase in fluorescence when bound to calcium ions

 Radiometric – easy to correct for photobleaching

 Can be used to accurately measure calcium ions

 Problems are it is UV exposure and the dual excitation is slow and requires specialised imaging equipment

GcAMP

GcAMP – genetic Ca2+ indicator

 Based on GFP, calmodulin and M13

 Single excitation, single emission

 Only fluoresces when bound to calcium ions

 Large increase in fluorescence when bound to calcium ions