* Embedded in the plasma membrane of post-synaptic cell
* Two classes of neurotransmitter receptors–
* receptors that are ion channels themselves (**ionotropic** or 'ligand-gated' ion channel)
* receptors that interface with separate ion channels (**metabotropic**, or G-protein coupled receptors)
* Ultimately, the binding of neurotransmitter results in the opening of ion channels and ion flux. This leads to either depolarization or hyperpolarization of the membrane potential depending on the **types of ions** flowing through the channel pores and the ions' respective **electrochemical driving forces**
Today we will dive a bit deeper into the structure and function of neurotransmitter receptors... last time was a warm up
For synaptic transmission, neurotrans 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...
In either case, neurotransmitter 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 flowwing through the channels and their respective electrical/chemical driving forces (Nernst)
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.
* 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
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.
<div><figcaption class="big">Patch clamp recording of current through single nAChR. Channels open for varying amounts of time while ACh is bound.</figcaption><img src="figs/Neuroscience5e-Fig-05.17-1R_copy_d0b6a64.jpg" height="500px"><figcaption>Neuroscience 5e Fig. 5.17</figcaption></div>
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
<div><figcaption class="big">end plate currents in a voltage-clamped muscle cell</figcaption><img src="figs/Neuroscience5e-Fig-05.17-2R_copy_fe44356.jpg" width="400px"><figcaption>Neuroscience 5e Fig. 5.17</figcaption></div>
<div>
<figcaption class="big">
depolarizing end plate potential recorded in muscle cell due
to the inward end plate currents
</figcaption><img src="figs/Neuroscience5e-Fig-05.17-1_copy_fd2d12e.jpg" width="400px"><figcaption>Neuroscience 5e Fig. 5.17</figcaption></div>
Note:
Indeed imagine we are doing an experiment where we stimulate a motor neuron and we record end plate currents in a muscle cell...
...then these 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 this panel on the right shows postsynaptic potential change or end plate potential produced by the EPC as we discussed previously
As we will learn in a few minutes, 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.
* - >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.
* Recall from 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 *E<sub>x</sub>*)
* Thus if one measured the ACh dependent current flow at different potentials, one could determine the membrane potential (*V<sub>m</sub>*) where current is 0. This is called the **reversal potential** or *E<sub>rev</sub>*
* The end plate current (EPC) at the muscle cell must therefore be *I<sub>ACh</sub>* and is equal to the driving force on an ion multiplied by its permeability (remember Ohm's law: *I = gV*)
* Predicts that current will be inward at potentials more negative than *E<sub>rev</sub>*, becomes small at potentials approaching *E<sub>rev</sub>*, and then becomes outward at potentials more positive then *E<sub>rev</sub>*
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 E<sub>rev</sub>.
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 V<sub>m</sub> and E<sub>rev</sub>) multiplied by the permeability for ACh gAch.
* Predicts that current will be negative (inward) at potentials more negative than E<sub>rev</sub>, becomes small at potentials approaching E<sub>rev</sub>, and becomes positive (outward) at potentials more positive then E<sub>rev</sub>.
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
<figure><figcaption class="big">Current-voltage relationships for different ion selectivities</figcaption><img src="figs/Neuroscience3e-2001-hypothetical-IV_copy_a3bfde0.jpg" height="400px"><figcaption>Neuroscience 2e 2001</figcaption></figure>
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.
*Ca2+ ions flow through CaV channels at a rate of ~106 ionss−1, but Na+ conductance is 500fold less through CaV channels*
*extracellular [Na+] is nearly 70fold higher than Na+, thus Ca2+ selectivity is crucial*
*Ca2+ and Na+ have nearly identical diameters (~2Å)*
*Ca2+ selectivity 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
---
## Influence of the postsynaptic V<sub>m</sub> on end plate currents
<figure><figcaption class="big">Effect of V<sub>m</sub> on postsynaptic muscle fiber end plate currents</figcaption><img src="figs/Neuroscience5e-Fig-05.18-2R_copy_33e27e0.jpg" width="700px"><figcaption>Neuroscience 5e Fig. 5.18, Takeuchi J Physiol 1960</figcaption></figure>
Note:
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?
---
## Influence of the postsynaptic V<sub>m</sub> on end plate currents
<div style="width:500px"><figcaption class="big">Expected E<sub>rev</sub> if nAChR permeable only to K⁺, Cl⁻, or Na⁺</figcaption><img src="figs/Neuroscience5e-Fig-05.18-4R_copy_a97bfef.jpg" width="300px"><figcaption>Neuroscience 5e Fig. 5.18</figcaption></div>
<div><figcaption class="big">Observed E<sub>rev</sub> is in between E<sub>k</sub> and E<sub>Na</sub></figcaption><img src="figs/Neuroscience5e-Fig-05.18-3R_copy_3d4e047.jpg" width="300px"><figcaption>Neuroscience 5e Fig. 5.18, Takeuchi J Physiol 1960</figcaption></div>
Note:
[#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
---
## Influence of the postsynaptic V<sub>m</sub> on end plate currents
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 E<sub>rev</sub> to the left and and raising the external [K] shifts E<sub>rev</sub> to the right.
* 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 E<sub>rev</sub> = 0
* E<sub>rev</sub> 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 (E<sub>rev</sub>) therefore normally at rest Na⁺ rushes in with very little K⁺ rushing out
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**
<figure><figcaption class="big">EPC: inward or outward; EPP: depolarizing or hyperpolarizing</figcaption><img src="figs/Neuroscience5e-Fig-05.20-2R_copy_f8c6010.jpg" width="700px"><figcaption>Neuroscience 5e Fig. 5.20</figcaption></figure>
Here is the key: you get inward currents at potentials more negative the E<sub>rev</sub> and you get outward currents at potentials more positive than E<sub>rev</sub>.
The resulting EPPs depolarize postsynaptic cell at potentials more negative than E<sub>rev</sub> and potentials more positive than E<sub>rev</sub> 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*
* 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 V<sub>m</sub> during the EPP approaches E<sub>rev</sub>, outward K⁺ flux is equal to inward Na⁺ flux. Therefore if the nACh receptor is open long enough, it will drive V<sub>m</sub> to E<sub>rev</sub>.
* If E<sub>rev</sub> is above action potential threshold, the probability of an action potential occurring is increased
* If E<sub>rev</sub> is below action potential threshold, the probability of an action potential occurring decreased
>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
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.
<!-- This plot shows two pretend neurotransmitters D and H that can depolarize or hyperpolarize the cell and their corresponding E<sub>rev</sub>s. This one causes an EPSP and inward current from Vrest, whereas this one causes an IPSP and an outward current from Vrest. -->
* 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 V<sub>m</sub> 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.
So imagine an experiment like we were doing before...
---
## IPSP type 1
* Here is an IPSP mediated by GABA activating Cl⁻ selective channels
* The reversal potential for the Cl current is negative to the resting potential and threshold
* Activation of Cl channels hyperpolarizes the neuron
<figure><figcaption class="big">IPSP mediated by Cl⁻ selective ion channel</figcaption><img src="figs/Neuroscience5e-Fig-05.21-0_2_copy_701a6c6.jpg" height="300px"><figcaption>Neuroscience 5e Fig. 5.21</figcaption></figure>
<div><figcaption class="big">IPSP mediated by Cl⁻ selective ion channel</figcaption><img src="figs/Neuroscience5e-Fig-05.21-0_3_copy_4d138b3.jpg" height="300px"><figcaption>Neuroscience 5e Fig. 5.21</figcaption></div>
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 E<sub>rev</sub> for the neurotransmitter receptor is more positive than threshold than it is excitatory. If it is more negative than threshold than it is inhibitory.
*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).
* 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
* A neuron integrates all this information and either fires a spike or not
* 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
As we’ve shown in our examples earlier the nAChR receptor is a non-selective cation channel. Or another way to think of it is that it is selective for cations.
>some subtypes of nAChR in the brain (those containing the b2 subunit) are located diffusely throughout the membrane of the neuron, with no obvious concentration at the synaptic junction (Hill et al. 1993).
a number of alpha and beta subunits have expression throughout brain (medulla, superior colliculus, cortex, beta2 subunit expression 'very high' in thalamus). Only alpha3 KO mice have high mortality [#Picciotto:2000].
[#Picciotto:2000]: Picciotto, M. R., Caldarone, B. J., King, S. L., and Zachariou, V. (2000). Nicotinic receptors in the brain. Links between molecular biology and behavior, Neuropsychopharmacology, 22(5), 451-65. PMID 10731620
Low (nM) concentrations of nicotine are found in the blood of moderate smokers (Henningfield et al. 1983). These are sufficient to enhance excitatory transmission in cultures of neurons from the medial habenula or the hippocampus (Gray et al. 1996; McGehee et al. 1995) [#Picciotto:2000]
Many effects of nicotine probably through presynaptic or preterminal nAChRs instead of through postsynaptic AChRs (Léna et al. 1993; Marshall et al. 1997; McGe- hee et al. 1995; Summers and Giacobini 1995; Vidal and Changeux 1993; Wonnacott et al. 1990; Yang et al. 1996) [#Picciotto:2000]
* In muscles the receptor has 2α, β, δ, γ, ε subunits. The α subunits bind ACh, both need to be bound for channel to open. α subunits also binds bungarotoxin and nicotine
*Also found in ganglia of PNS. Mediate peripheral cholinergic responses of autonomic effector organs like heart, smooth muscle, exocrine glands. Inhibition of heart rate by vagus nerve.*
* Kainate receptors, or KARs, are ionotropic receptors that respond to the neurotransmitter glutamate.
* Kainic acid (kainate) is a natural marine acid present in some seaweed. Kainic acid is a potent neuroexcitatory amino acid that acts by activating receptors for glutamate,
* Glutamate receptors that allow flow of Ca²⁺ as well as Na⁺ and K⁺. As a result EPSPs produced by NMDA receptors can increase the Ca²⁺ concentration in the neuron. Acts as a second messenger to activate cellular processes
* Formed as a heterotetramer of 4 subunits (typically 2 NR1 and 2 NR2 subunits)
* Blocked by Mg²⁺ in the pore during hyperpolarizing conditions. Depolarization can remove block. Needs either a bunch of presynaptic cells to fire at the same time or repeated firing of presynaptic cell to open channel
* Key component of a model for learning
* Evoke EPSPs that are slow and long lasting
* PCP “angel dust” binds and clogs channel. Get symptoms similar to schizophrenia
* group I (mGluR1, mGluR5) associated with IP3 signaling and ER Ca2+ channel opening. Also associated with Na+ and K+ channels. Can result in EPSPs but can also result in IPSPs.
* activated selectively by 3,5-dihydroxyphenylglycine (DHPG) (but not other groups)
* group II mGluRs 2 and 3 prevent formation of cAMP (by activating Gi that inhibits adenylyl cyclase) and result in presynaptic inhibition (not apparently affecting PSPs directly)
* group III, including mGluRs 4, 6, 7, and 8 prevent formation of cAMP and have similar functional pathway and consequences as group II
* A and C are inhibitory because their channels are permeable to Cl⁻. The flow of Cl⁻ into the cell lowers the potential. E<sub>rev</sub> is less than the threshold potential
* Pentamers, subunit diversity as well as variable stoichiometry, allows for variable functions of GABA receptors
* Glycine receptors generally have the same properties as GABA receptors
* GABAB metabotropic receptors always inhibitory. Coupled indirectly to K+ channels and can decreased Ca2+ conductance resulting in less cAMP production. Baclofen is a potent and selective GABAB agonist. GABA responses that are insensitive to bicuculline and baclofen are termed GABAC responses.
* GABAA: muscimol potent agonist from mushrooms. Bicuculline classical antagonist and convulsant.
>Found primarily in the fruit of the climbing plant Anamirta cocculus, it has a strong physiological action. It acts as a non-competitive channel blocker for the GABAA receptor chloride channels.[3] It is therefore a channel blocker rather than a receptor antagonist.
<figure><figcaption class="big">Erev is at the Nernst potential for Cl⁻ (e.g. –80 mV)</figcaption><img src="figs/Coombs-JPhysiol1955-Fig1_copy_1932d79.jpg" height="400px"><figcaption>Coombs et al., J Physiol 1955 Fig. 1</figcaption></figure>
Coombs, Eccles, Fatt 1955: double barreled pipete, inject small currents through one barrel (for voltage clamp) in biceps motorneuron (crustacean) to hold Vm while stimulating afferent nerve inputs to get IPSPs. Erev was found to be close to ECl. Notice hyperpolarization when Vm was above -78 mV, small depolarizations when Vm below -80mV. They found that messing with Cl- concentrations would correspondingly alter the IPSPs but not when messing with Na or K concentrations. Thus Cl- ion flux is necessary for the IPSPs.
[#Coombs:1955]: Coombs, J. S., Eccles, J. C., and Fatt, P. (1955). The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential, J Physiol, 130(2), 326-74. PMID 13278905
<figure><figcaption>Stimulate GABA producing interneuron, record from post-synaptic neuron</figcaption><img src="figs/Neuroscience5e-Fig-06.09-2R_9a77707.jpg" height="300px"><figcaption>Neuroscience 5e Fig. 6.9</figcaption></figure>
Chavas and Marty performed Gramacidin perforated patch recordings from young rat cerebellum interneurons and purkinje cells. *Interneurons had more depolarized GABAA reversal potentials than purkinje cells at matched ages (e.g. P12, likely from higher [Cl-]intra for interneurons compared to purkinje cells).*
[#Chavas:2003]: Chavas, J. and Marty, A. (2003). Coexistence of excitatory and inhibitory GABA synapses in the cerebellar interneuron network, J Neurosci, 23(6), 2019-31. PMID 12657660
>GABA’s effect is to reduce neural activity by allowing chloride ions to enter the post-synaptic neuron. These ions have a negative electrical charge, which helps to make the neuron less excitable. This physiological effect is amplified when alcohol binds to the GABA receptor, probably because it enables the ion channel to stay open longer and thus let more Cl⁻ ions into the cell.
>Still other substances block a natural neuromediator. Alcohol, for example, blocks the NMDA receptors.
>It has now been established that all substances that trigger dependencies in human beings increase the release of a neuromediator, dopamine, in a specific area of the brain: the nucleus accumbens.
Opioid peptides distributed throughout the brain. Colocalize with GABA and 5-HT. Tend to be depressants. They act like analgesics when injected intracerebrally. Initiate effects through GPCRs. Activate at low concentrations (nM to uM). mu, delta, kappa opioid receptor subtypes play role in reward and addiction. mu-receptor is primary site for opiate drugs.
Receptors for ATP and adenosine are widely distributed through the nervous system as well as other tissues.
One class of purinergic receptors for ATP and adenoscie are P2X-receptors which are ionotropic non-selective cation receptors. Others are GPCRs like A2A adenosine receptor throughout brain and heart, adipose tissue, and kidney. Xanthines like caffeine and theophylline block adenosine receptors and this is thought to be the cause of its stimulant effects.
* Neurotransmitter receptors bind neurotransmitters. Tremendous diversity but with commonalities
* Two types– ionotropic (ligand-gated ion channel) and metabotropic (G-protein coupled receptor)
* Both types lead to opening or closing of ion channels. These conductance changes can either increase or decrease the probability of firing an action potential
* Because postsynaptic neurons are usually innervated by many different inputs, it is the combination of EPSP and IPSPs that determines whether a cell fires and if an action potential occurs
