* Embedded in the plasma membrane of post-synaptic cell.
* Either are ion channels themselves (ionotropic, or ligand-gated ion channel) or interface with ion channels (metabotropic, or G-protein coupled receptors).
* Ultimately, the binding of neurotransmitter causes opening of channels and ion flux. This can lead to depolarization or hyperpolarization of the membrane potential depending on the ion concentrations and the particular ion species flowing in or out.
Note:
Today we will dive a bit deeper into the structure and function of neurotransmitter receptors.
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 nACh receptor 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 potential of the muscle, the EPP, which can trigger an action potential.
Note:
nACh Receptors are ionotropic receptors 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.
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## Outside-out patch clamping showing ACh gated currents
Neuroscience 5e 5.17
Channels open for various amounts of time at a given potential.
The binding of a neuro-transmitter to its receptor usually opens (sometimes closes) ion channels.
The figure shows a simple case. In the absence of ACh, the channel is closed. In the presence of high ACh (the channel always has ACh bound), the channel opens and closes. These repeated breif openings are seen as downward deflections corresponding to inward current.
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## Activation of nACh receptors at neuromuscular synapses
post-synaptic muscle cell voltage clamped to look at currents
not voltage clamped, inward EPC causes depolarizing EPP in the muscle cell
The traces show inward currents through these ionotropic ACh channels, showing the currents stemming from a single channel, 10 channels, and a million channels.
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.
* acetylcholine causes opening of a cation channel in the receptor capable of transmitting 15,000–30,000 Na+ or K+ ions a millisecond
* - >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.
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## How do we figure out what ions flow through the nACh receptor?
* From Nernst equation– the equilibrium potential of a cell is the potential at which there is a balance between the concentration gradient and the electrochemical gradient.
* In other words– 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 potential that current flow was 0. This is called the reversal potential or Erev.
* The end plate current (EPC) is therefore IACh and is equal to the driving force on an ion multiplied by its permeability (remember Ohms 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.
Note:
Use 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.
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## Influence of the postsynaptic membrane potential on end plate currents
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.
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## Hypothetical ion channel selectivities and the reversal potential
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) than
11Na, 12Mg, 17Cl, 19K, 20Ca
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## 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.
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## Influence of the postsynaptic membrane potential 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 Erev to the left and and raising the external [K] shifts Erev to the right.
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.
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.
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## Na+ and K+ movements during EPCs and EPPs
* 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
* At normal resting potentials as the nACh receptor opens, many Na+ ions rush in and a few K+ rush out. This causes the cell to depolarize. As the potential goes toward Erev, as many K+ go out as Na+ goes in. Therefore the nACh receptor if open long enough would drive the potential to Erev. If Erev is above threshold, the probability of an action potential happening is increased and is called an excitatory postsynaptic potential (EPSP)
* If Erev is below threshold the probability of an action potential is decreased. Called an inhibitory postsynaptic potential (IPSP).
>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.
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## Postsynaptic potentials between neurons
* Excitatory postsynaptic potentials (EPSP) increases the likelihood that an action potential will be initiated in the post synaptic cell.
* Inhibitory postsynaptic potentials (IPSP) decreases the likelihood that an action potential will be initiated in the post synaptic cell.
In fact we can generalize the properties that we’ve learned about EPCs through ionotropic AChR and their effects on EPPs at the neuromuscular junction to the general case of chemical synapses between pairs of neurons.
Instead of so called EPPs, the postsynaptic potentials between neurons we call excitatory if it increases the likelihood of an AP firing in a postsynaptic cell and inhibitory 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 Erevs. This one causes an EPSP and inward current from Vrest, whereas this one causes an IPSP and an outward current from Vrest.
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## Similar mechanisms exist at all chemical synapses
* Instead of end plate current called postsynaptic current (PSC).
* Instead of end plate potential called post synaptic potential (PSP).
* excitatory PSP– EPSP increases likelihood of an action potential
* inhibitory PSP– IPSP decreases likelihood of an action potential
Note:
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## EPSP summation
* Unlike the neuromuscular junction– at synapses between neurons an EPSP is usually not very strong, usually well below threshold. What is needed is a bunch of EPSPs to sum together. Typically neurons receive more than a thousand synapses. It is basically the summation of EPSPs and IPSPs that determine whether or not an action potential occurs. If sum is above threshold, an AP happens.
Note:
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## EPSP
* Here is an EPSP mediated by glutamate activating nonselective cation channels.
* 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.
ECl
Neuroscience 5e 5.21
Time (ms)
Note:
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## IPSP #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.
ECl
Neuroscience 5e 5.21
Time (ms)
Note:
Also called shunting inhibition. Na+ channels persistently in state of inactivation due to small depolarizing pulses.
So just remember 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 demon- strated 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 cotrans- porter (NKCC) and K -Cl cotransporter (KCC). NKCC1 im- ports 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).
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## Remember in neurons an EPSP is not driven from one pulse
* Activation of ionotropic receptors opens nonselective cation channels.
* The first stimulation does not reach threshold.
* More intense stimulation yields a larger EPSP and an AP.
* 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.
Note:
Of course we are greatly simplifying everything here, a single neuron may have as many as 10K synaptic inputs.
* 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.
Note:
some neurons in the globus pallidus have voltage gated Na channels.
g protein subunits or intracellular messengers modulate ion channels
ion channel opens
ions flow across membrane
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## Cholinergic receptors
* Best studied– the nicotinic ACh receptor (nAChR)
* Pentamer-5 subunits to make a pore. Selective for cations.
* Nicotine can mimic ACh to stimulate receptor, this is called an agonist. Most effects of nicotine go through this receptor. Nicotine is not cleared very well so receptor stays open longer which leads to larger EPSPs
* nACh receptors produce EPSPs.
* Many toxins specifically bind to and block nicotinic receptors called antagonists.
* alpha-bungarotoxin (snake venom)– binds to alpha subunit of nAChR very tightly and prevents ACh from activating it.
Note:
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.
5 subunits
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## nAChR
* Green is motor axons, red is where Bungarotoxin binds, defines the endplates
* In muscles the receptor has 2 α, β,γ,ε subunits. The α subunits bind ACh, both need to be bound for channel to open. α subunits also binds bungarotoxin and nicotine.
* Multiple isoforms for each subunit, depending on which isoform is in channel get different properties
* In neurons its slightly different. 5 subunits 3α:2β. Bungarotoxin only inhibits muscle nACh receptors.
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.
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## Muscarinic ACh receptors
* Muscarine, a poisonous mushroom alkaloid, is an agonist.
* Metabotropic, mediates most of ACh effects in the brain.
* 5 or so isoforms
* mACh blockers are used for pupil dilation (atropine), motion sickness (scopolamine) and asthma treatment (ipratropium).
* [Also used for bad things http://www.rense.com/general38/frug.htm](http://www.rense.com/general38/frug.htm)
Note:
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## Glutamate receptors
* Both ionotropic and metabotropic
* Ionotropic– NMDA receptors, AMPA receptors, and Kainate receptors (named after the agonists that stimulate them).
* All are non-selective ion channels with Erev close to 0 (above threshold therefore excitatory).
* Formed from an association of many subunits, that can combine to create many isoforms.
Note:
tetramers
3 classes, 8 subunits
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,
* Domoic acid is a structural analog of kainic acid and proline.
* Domoic acid (DA) is a kainic acid analog neurotoxin that causes amnesic shellfish poisoning
* 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.
* Needs a co-agonist, glycine to open channel
* 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.
Note:
PCP “angel dust” binds and clogs channel. Get symptoms similar to schizophrenia. Some hypothesize NMDA receptor is involved in this disease.
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## NMDA receptor currents requires glutamate, glycine, and removal of voltage-gated Mg2+ block
* Glycine is a co-agonist-no glycine no current.
* Mg2+ blocks pore-is removed by depolarization.
* This can happen because AMPA and NMDA receptors are often in the same synapse.
* Often leads to inhibition of postsynaptic Ca2+ and Na+ channels
* But sometimes inhibitory sometimes excitatory
Note:
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## GABA receptors
* Three types of GABA receptors: A, B and C.
* A and C are ionotropic, B is metabotropic.
* A and C are inhibitory because their channels are permeable to Cl-. The flow of Cl- into the cell lowers the potential. Erev is less than the threshold potential.
* Pentamers, subunit diversity as well as variable stoichiometry, allows for variable functions of GABA receptors.
* Glycine receptors have generally the same properties as GABA receptors
>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.
>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.
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## Serotonin receptors
* Large family of receptors called 5-HT 1-7.
* 5-HT3 is a ligand gated non-selective cation channel, thus it is excitatory.
* Same basic structure as nACh receptor.
* All others are metabotropic– likely that perturbations in these receptors are involved in many neural disorders.
Note:
most receptors are metabotropic
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## Catecholamine receptors
* Act exclusively by activating G-protein coupled receptors. Contribute to complex behaviors.
* Norepinephrine and epinephrine each act on α and β adrenergic receptors.
* Mostly used to control smooth muscles, especially cardiovascular.
* B-blockers are used to treat hypertension, anxiety, and panic.
Note:
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## Peptide receptors
* Virtually all mediate their effects by activating G-protein coupled receptors.
* Neuropeptide-Y receptor important in food intake/ obesity.
* Opiate receptors have been identified and shown to be important in addiction (e.g. µ-opioid receptor).
Note:
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.
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## ATP and other purines (adenosine)
* ATP is contained in all synaptic vesicles
* Has specific receptors on post-synaptic cells
* P2X
* A2A adenosine receptor (blocked by caffeine)
* Generally excitatory in nature
* Used in spinal cord, motor neurons, and other ganglia.
Note:
Another neurotransmitter that we didn’t talk about last time is
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.
