* Neurotransmitter receptors are embedded in the plasma membrane of the post-synaptic cell and are always one of the following:
1. ion channels (**ionotropic** or 'ligand-gated' ion channel)
2. 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**
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*).
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...
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.
* Or guanylyl cyclase (GTP->cGMP) --> Protein kinase G etc.
* All G-protein receptor activations lead to downstream second messsaging (cAMP, diacylglycerol, IP3) --> protein kinases, Ca2+ --> leading to phosphorylation state changes including... ion channels
* Three amplification steps here! (receptor production of G proteins, adenylyl cyclase production of cAMP, protein kinase substrate phosphorylation). Source signal amplification.
* 3% of our genome is codes for protein phosphorlation state genes (500 protein kinases and 200 protein phosphatases)
* cAMP dependent protein kinases (PKA)
* Ca^2+^ - calmodulin depedent protein kinase type II (CaMKII predominant in neurons, most abundant protein component of the post synaptic density)
* Protein kinase C (PKC)- activated by Ca^2+ (moves PKC from cytosol to membrane) and diacylglycerol (DAG) and then phosphorylates substrates
* 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.
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.
<div><figcaption class="big" style="width:500px">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>
...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.
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.
>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
* 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 there is no net ion flux (*I<sub>x</sub>* = 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* [#Tang:2014]
*extracellular [Na+] is nearly 70fold higher than Ca2+, thus Ca2+ selectivity is crucial* [#Tang:2014]
*Ca2+ and Na+ have nearly identical diameters (~2Å)* 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
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?
<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
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.
* 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
</div>
<div style="float:left;margin:0 15px;"><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></div>
<div style="width:400px; float:left"><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).
* Not so simple-- synaptic inputs can be summed in space and time within a neuron
* Recall a single neuron may have as many as 10,000 different synapses. Some are excitatory some inhibitory, some strong some weak. Some at the tips of dendrites, some near the cell body
* Integration of all these little postsynaptic bioelectric waves determines whether the neuron fires an action potential
* In many neurons the decision to initiate an action potential is at the axon hillock. Contains a high density of voltage dependent Na^+^ channels and is contains membrane with lowest threshold
* This is due graded potentials that spread passively
* Temporal summation, process by which consecutive synaptic potentials at the same site are added together.
* Spatial structure of the 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
* Different synapses will have different time constants
* Some dendrites have voltage gated Na^+^ channels (albeit lower density than axons), these can amplify inputs
* 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
Time constant
: time needed for for resistive current (I~r~, current due to ions flowing through channels) and membrane potential (V~m~) to reach **63%** of their *asymptotic values* is proportional to the combination of resistance and capacitance of the circuit in question (across the cell membrane)
: membrane current (I~m~) is sum of I~r~ and the capacitive current (I~c~)
: I~m~ = I~r~ + I~c~
: capacitance of membrane: during change in applied voltage or current across membrane, positively charged ions pile on surface of one side of membrane and **electrostatically** interact with cations on the other side of membrane surface (membrane acts as thin impermeable surfaces in parallel, like a capacitor), repeling them and inducing immediate, fast capacitive current along membrane
: capacitive current falls with an exponential time course. And the membrane potential rises with **same exponential** time course
: Relation of membrane potential at time *t* during charging of capacitance is given by V~t~ = V~inf~(1 - *e*^-t/RC^), where V~inf~ is the membrane potential at an infinite asymptotic value of the exponential curve. When t = RC, then we have V~t~ = V~inf~ ( 1 - *e*^-1^) ==> V~inf~ (0.63)
