26 KiB
Neurotransmitter receptors
- Neurotransmitter receptors are embedded in the plasma membrane of the post-synaptic cell and are always one of the following:
- ion channels (ionotropic or 'ligand-gated' ion channel)
- receptors that interface with separate ion channels (metabotropic, or G-protein coupled receptors)
- Neurotransmitter receptor activation following ligand (neurotransmitter) binding results in the opening of ion channels and ionic flux. This ion flux is the postsynaptic current (or 'end plate' current for a muscle cell)
- These postsynaptic currents result in depolarization or hyperpolarization of the membrane potential (postsynaptic potential or 'end plate' potential) depending on the types of ions flowing through the channel pores and the ions' respective electro-chemical driving forces
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Diving a bit deeper into the structure and function of neurotransmitter (NT) receptors now...
For synaptic transmission, NT receps are generally located in the post-synaptic membrane (though there are exceptions, e.g. some transmitter receptors may be located on pre-synaptic membrane or at non synaptic site in the cell).
Two classes of NT receptors.
In either case, NT binding will result in ion channels opening and ion flux across the post-synaptic membrane. Whether this results in hyperpolarization or depolarization of the membrane will be due to the types of ions flowing through the channels and their respective electrical/chemical driving forces (Nernst)
Changing the postsynaptic membrane potential inturn affects the electrochemical driving forces regulating ion flux. So currents may change amplitude and direction during the course of a postsynaptic potential. Read on...
Ionotropic neurotransmitter receptors
- Neurotransmitter binds receptor
- Channel opens, allowing ions to flow through
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The ionotropic receptors are the ones you’ve probably seen in our synaptic diagrams so far, where NT binds directly to an ion channel pore, causing it to open and allow ions to move through the pore.
- neurotransmitter binds
- channel opens
- ions flow across membrane
Metabotropic neurotransmitter receptors
- G-protein coupled receptor signalling results in modulation of nearby ion channels for metabotropic receptors.
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Metabotropic transmitter receptors are G-protein coupled receptors, also known as seven-transmembrane domain receptors in you cell biology courses.
- neurotransmitter binds
- g protein binds and is activated
- g protein subunits or intracellular messengers modulate ion channels
- ion channel opens
- ions flow across membrane
Effector enzymes for activated G-proteins include adenylyl cyclase (ATP->cAMP), phopholipase C, guanylyl cyclase (GTP->cGMP) etc. Then downstream second messsaging (cAMP, diacylglycerol, IP3) --> protein kinases, Ca2+. And then more phosphorylation state changes.
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Neurotransmitter receptors video summary
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Nicotinic acetylcholine receptors (nAChR)
- Ionotropic receptor
- Acetylcholine (ACh) binds the nAChR– this opens the channel
- ACh causes nAChR to open transiently and stochastically (patch clamp studies)
- An action potential causes lots of ACh molecules to be released simultaneously, transiently opening many nACh receptors
- The summed current flow into the muscle cell is called the end plate current (EPC). Current flow changes the transmembrane potential of the muscle, the end plate potential (EPP), which triggers an action potential
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So to understand the properties of ionotropic neurotransmitter receptors lets start with the nicotinic ACh receptor (abbreviated nAChR).
nACh Receptors are ionotropic or ligand-gated receptors where the ligand is ACh and are the receptor you’ve heard the most thus far, being the one that underlies end plate currents at the neuromuscular junction that cause end plate potentials in muscle cells.
- stochastic
- having a random probability distribution or pattern that may be analyzed statistically but may not be predicted precisely
Patch clamping shows ACh gated currents through nicotinic ACh receptors
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The binding of a neurotransmitter to its receptor usually opens (sometimes closes) ion channels.
The figure shows a simple case. In the absence of ACh, the nAChR is closed. In the presence of high [ACh] (the channel always has ACh bound), the channel opens and closes. These repeated brief openings are seen as downward deflections corresponding to inward current. Notice the current amplitudes in this patch clamp trace below are unitary or quantal indicating that a single channel is being recorded in this case...
These look like microscopic currents you get in single channel patch clamp recordings like we discussed previously.
If this piece of membrane and channel is from a muscle cell than a bunch of these currents put together are the ones that give rise to the end plate potentials we for muscle cells before.
Activation of nAChR at neuromuscular synapses
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Imagine we are doing an experiment where we stimulate a motor neuron and we record end plate currents in a muscle cell...
...then the traces on the left show inward currents through these ionotropic ACh channels in the muscle cell, showing the currents stemming from a single channel, 10 channels, and hundreds of thousands of channels. Notice the amplitudes of the currents scale.
...and the panel on the right shows postsynaptic potential change or end plate potential produced by the EPC as we discussed previously
As we will learn shortly, the channel opened by ACh lets mostly Na⁺ through resulting in these inward currents that depolarize the muscle cell, resulting in EPPs and typically resulting in APs as we’ve discussed before.
from http://www.ncbi.nlm.nih.gov/books/NBK21586/:
Two factors greatly assisted in the characterization of the nicotinic acetylcholine receptor. First, this receptor can be rather easily purified from the electric organs of electric eels and electric rays; these organs are derived from stacks of muscle cells (minus the contractile proteins) and thus are richly endowed with this receptor. (In contrast, this receptor constitutes a minute fraction of the total membrane protein in most nerve and muscle tissues.) Second, α-bungarotoxin, a neurotoxin present in snake venom, binds specifically and irreversibly to nicotinic acetylcholine receptors.
- acetylcholine causes opening of a cation channel in the receptor capable of transmitting 15,000 – 30,000 Na⁺ or K⁺ ions a millisecond
What ions flow through the nicotinic ACh receptor?
- Nernst equation– the equilibrium potential of a cell for ion x is the potential at which the electrochemical driving forces is balanced for ion x (i.e there is no net flow of ion x at the equilibrium potential Ex)
- Thus if one measured the ACh dependent current flow at different potentials, one could determine the membrane potential (Vm) where there is no net ion flux (Ix = 0). This is called the reversal potential or Erev
- The end plate current (EPC) at the muscle cell must therefore be IACh and is equal to the driving force on an ion multiplied by its permeability (remember Ohm's law: I = gV)
- IACh = gACh(Vm – Erev)
- Predicts that current will be inward at potentials more negative than Erev, becomes small at potentials approaching Erev, and then becomes outward at potentials more positive then Erev
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Now using our good friend the Nernst eqn, which you can recall is…
Since we know there isn’t any net flow of an ion x, at the Ex, we can measure the ACh dependent currents at different potentials and figure out the potentials at which current flow is 0.
When we are talking about the potential at which postsynaptic currents like the endplate current reverses from inward net ion flux to outward net ion flux, we call this potential the reversal potential denoted Erev.
We can call the endplate current then the IAch or the current flowing through the ACh receptor at skeletal muscle endplate membrane and IAch is therefore equal to the driving force (which is the difference between Vm and Erev) multiplied by the permeability for ACh gAch.
This would then predict that current will be inward at potentials more negative than Erev…
- Predicts that current will be negative (inward) at potentials more negative than Erev, becomes small at potentials approaching Erev, and becomes positive (outward) at potentials more positive then Erev.
Measure postsynaptic (end plate) currents while stimulating motor neuron
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A postsynaptic muscle fiber is voltage clamped to control the muscle fiber’s membrane potential, while the presynaptic neuron is stimulated to cause ACh release at the end plate synapse.
Hypothetical ion channel selectivities and the reversal potential
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So let’s imaging what the current-voltage relationships would look like for different channel selectivities. Remember the reversal potential is when there there is no net ion flux, so it 0 nA on all these graphs and if a channel is selective to only K, it would be equal to the Ek.
If the channel was selective only to Na, than the Erev would be equal to ENa. Same for chloride.
If the channel was a non-selective cation channel (passing both K and Na) then the current-voltage relationship would look like...
11Na, 12Mg, 17Cl, 19K, 20Ca
Ca2+ ions flow through CaV channels at a rate of ~106 ions s−1, but Na+ conductance is 500fold less through CaV channels [#Tang:2014] extracellular [Na+] is nearly 70fold higher than Ca2+, thus Ca2+ selectivity is crucial [#Tang:2014] Ca2+ and Na+ have nearly identical diameters (~2 Å) 1 Å = 100 pm (Ca2+ larger atomic size, but Na+ has larger ionic size|hydration shell). Ca2+ selectivity is from high affinity binding, preventing Na+ permeability. Multi site pore, with knock on mechanism to push Ca2+ ions through [#Tang:2014]
[#Tang:2014]: Tang, L., Gamal El-Din, T. M., Payandeh, J., Martinez, G. Q., Heard, T. M., Scheuer, T., Zheng, N., and Catterall, W. A. (2014). Structural basis for Ca2+ selectivity of a voltage-gated calcium channel, Nature, 505(7481), 56-61. PMID 24270805
Postsynaptic Vm affects the magnitude and direction of end plate currents
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These little transients are just stimulus artifacts, but look at the postsynaptic end plate currents in these at these different Vms. Look what happens when Vm is at 0mV, there is no current and then above 0 mV it flips from being inward to net outward current...
We already know that ACh is essential for the end plate currents-- therefore we can say that this EPC is IAch. Therefore what is the Erev for IAch?
Postsynaptic Vm affects the magnitude and direction of end plate currents
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[#Takeuchi:1960]: Takeuchi, A. and Takeuchi, N. (1960). On the permeability of end-plate membrane during the action of transmitter, J Physiol, 154(), 52-67. PMID 13774972
Shifting ENa+ or EK+ shifts Erev of the neuromuscular endplate current
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So it seems that the ACh activated ion channels are equally permeable to Na and K and this was tested in 1960 by Akira and Noriko Takeuchi by changing the extracellular concentration of these ions. As predicted, lowering [Na] shifts Erev to the left and and raising the external [K] shifts Erev to the right.
What ions flow through the nACh receptor?
- Voltage clamping experiments show that there are large inward currents at -110 mV, smaller currents at -60 mV and no current at 0 mV. Outward currents at +70 mV. Therefore Erev = 0
- Erev is not at any of the equilibrium potentials for a single ion, lies in between K⁺ (-100 mV) and Na⁺ (+70 mV)
- Altering the K⁺ concentration or the Na⁺ concentration will change the membrane potential. Therefore both Na⁺ and K⁺ are permeable through the nACh receptor
- nACh receptor can conduct both Na⁺ and K⁺ ions. The direction of flow is dependent on the membrane potential. The normal resting state of muscle is -100 mV, well below 0 mV (Erev) therefore normally at rest Na⁺ rushes in with very little K⁺ rushing out
Note:
As we will see in a minute voltage clamp experiments show that there is a…
Erev…
Furthermore, altering…
Therefore we can conclude that the nAChR can conduct both Na and K ions.
Na⁺ and K⁺ movements during EPCs and EPPs
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Even though these ionotropic channels opened by ACh are permeable to both Na and K, at the resting membrane potential the EPC is generated primarily by Na influx because of the reduced driving force on K since at Vrest the membrane potential is closer to Ek.
In fact the Na⁺ and K⁺ permeabilities of the nAChR channel are similar, therefore the magnitudes of the Na⁺ and K⁺ currents depends on the driving forces present for each ion
Na⁺ and K⁺ movements during EPCs and EPPs
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Here is the key: you get inward currents at potentials more negative the Erev and you get outward currents at potentials more positive than Erev.
The resulting EPPs depolarize postsynaptic cell at potentials more negative than Erev and potentials more positive than Erev hyperpolarize the cell.
Since the Na⁺ and K⁺ permeabilities of this channel are similar, the magnitudes of the Na⁺ and K⁺ currents depends on the driving forces present for each ion
nAChR summary
- When the nAChR opens at normal resting potentials many Na⁺ ions rush in and a few K⁺ rush out. This causes a depolarizing EPP in the muscle cell. As the Vm during the EPP approaches Erev, outward K⁺ flux is equal to inward Na⁺ flux. Therefore if the nACh receptor is open long enough, it will drive Vm to Erev.
- If Erev is above action potential threshold, the probability of an action potential occurring is increased
- If Erev is below action potential threshold, the probability of an action potential occurring decreased
Note:
http://www.nature.com/nrd/journal/v1/n6/full/nrd821.html:
In the case of this modified muscle nAChR, the conductance of the pore is sensitive to the presence of negative charge at three locations that form three negatively charged rings in and near the M2 domain56. So, intensive studies of the M2 segment have been carried out to determine the amino acids that are responsible for the cationic or anionic selectivity of receptors.
Similar mechanisms exist at all chemical synapses
For synapses between neurons:
- Postsynaptic current (PSC) is similar to an end plate current
- Post synaptic potential (PSP) is similar to an end plate potential
- Excitatory PSP (EPSP)– increases likelihood of an action potential occurring
- Inhibitory PSP (IPSP)– decreases likelihood of an action potential occurring
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So now let's generalize the properties that we’ve learned about EPCs through ionotropic AChR and their effects on EPPs at the neuromuscular junction to the case of chemical synapses between any pair of neurons...
But instead of the so called EPPs, we'll call the postsynaptic potentials between neurons we call excitatory PSP if it increases the likelihood of an AP firing in a postsynaptic cell and inhibitory PSP if it decr the probability of an AP occurring in a postsynaptic cell.
EPSP summation
- Unlike the neuromuscular junction– at synapses between neurons an individual EPSP is usually not very strong, typically well below threshold.
- Multiple EPSPs need to be summed together for the neuron's Vm to reach threshold. Individual neurons can receive thousands synapses. It's the summation of EPSPs and IPSPs that determine whether or not an action potential occurs.
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Excitatory postsynaptic potential (IPSP)
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Imagine an experiment like the endplate potental recordings at the neuromuscular junction before but this time on a neuron in the CNS
Inhibitory postsynaptic potential (IPSP) type 1
- An IPSP mediated by a GABA activated chloride selective channel that hyperpolarizes the neuron
- Reversal potential for the Cl⁻ current is negative to the resting potential and action potential threshold
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IPSP type 2
- The reversal potential for the Cl⁻ current is positive to the resting potential but negative to threshold
- Activation of Cl⁻ channels depolarizes the neuron. Stabilizes membrane potential below threshold
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Imagine if a separate EPSP input brought Vm of this neuron to -41 mV, just below -40mV threshold. Since this is now postive to the ECl of -50mV, further activity at the IPSP synapses will now hyperpolarize the neuron back towards -50mV.
This can also be called shunting inhibition. In this case Na⁺ channels could persistently be in a state of inactivation due to small ongoing depolarizing and hyperpolarzing pulses keeping the neurons Vm below threshold.
So just remember, the key is that if the Erev for the neurotransmitter receptor is more positive than threshold than it is excitatory. If it is more negative than threshold than it is inhibitory.
Blocking NKCC1 with bumetanide disrupts excitatory synapse development in the cortex
Bumetanide, a selective NKCC1 inhibitor, has been demonstrated to suppress certain forms of epileptiform activity in vitro and in vivo, presumably by attenuating the depolarizing effect of GABA (Dzhala et al., 2005; Kilb et al., 2007)
effect of GABA on membrane polarity depends on the Cl gradient created by the expression of Na -K -2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC). NKCC1 imports Cl and is expressed from the embryonic stage until the first postnatal week, whereas KCC2 exports Cl and is weakly expressed at birth and upregulated as the brain matures (Plotkin et al., 1997; Rivera et al., 1999; Li et al., 2002). The temporal expression patterns of these two transporters correspond to the switch of GABA from being excitatory to inhibitory during the first few weeks of rodent postnatal life (Delpire, 2000).
Summation of postsynaptic potentials
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Summation
- In general EPSPs in neurons are small 0.2–0.4 mV
- Most neurons are somewhere between 10–20 mV below threshold. If everything was linear that it would take the sum of 50 or so inputs to trigger AP
- Not so simple. Some inputs are bigger than others, the inputs can be summed differently– spatially or temporally
- A single neuron can have as many as 10,000 different synapses. Some excitatory some inhibitory, some strong some weak. Some at the tips of dendrites, some near the cell body
- Integration of all the postsynaptic potentials determines whether the neuron fires an action potential
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Of course we are greatly simplifying everything here, a single neuron may have as many as 10K synaptic inputs.
Neural integration
- How does a neuron integrate all the information it is getting?
- In most motor neurons and interneurons the decision to initiate an action potential is at the axon hillock. Contains a high density of voltage dependent Na⁺ channels. Contains membrane with lowest threshold
- Axon hillock is senses the local state of the cell, which is the combination of all the EPSPs and IPSPs going on at one time
- This is mostly due potentials that spread passively
- Temporal summation, process by which consecutive synaptic potentials at the same site are added together. Different synapses will have different time constants
- Length constant of the cell determines the degree to which a depolarization current decreases as it spreads passively. Easier to sum inputs on the same dendritic branch than on different branches
- Some dendrites even have voltage gated Na⁺ channels, these can amplify inputs
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Summation of postsynaptic potentials video
Midterm 1
mean 83.15
std 9.11
max 100
min 55
median 84
