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neurophysiology3.md
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@@ -36,7 +36,7 @@ Answer to myelinated question from last time:
>It seems to have arisen independently in evolution several times in vertebrates, annelids and crustacea.
>absent in primitive members of the vertebrate line (hagfish and lampreys)
>absent in primitive members of the vertebrate line (hagfish and lampreys)
>Myelin has not been reported in either molluscs or insects
@@ -91,8 +91,7 @@ Makes it easy to introduce things to the extracellular side of the channel
Note:
---
--
## The Nobel Prize in Physiology or Medicine (1991)
@@ -106,7 +105,7 @@ Erwin Neher
<div style="width:600px; float:left;"><img src="figs/sakmann_postcard_ad6439c.jpg" height="200px"><figcaption class="big">
Bert Sakmann
Bert Sakmann
</figcaption></div>
@@ -114,7 +113,7 @@ Bert Sakmann
Note:
Patch clamp established by E. Neher adn B. Sakmann at Max Planck Institute in Germany 1976.
---
@@ -159,6 +158,8 @@ Patch a piece of membrane and block K currents. Do a bunch of short recordings w
Transient channel opening in Na⁺ channels (inward current).
This research is from Bezanilla and Correa 1995, Vandenburg and Bezanilla 1991, Correa and Bezanilla 1994
---
## Measurements of ionic currents flowing through single Na⁺ channels
@@ -177,12 +178,14 @@ Transient channel opening in Na⁺ channels (inward current).
Note:
get something similar to this microscopic current shown at the top.
get something similar to this microscopic current shown at the top.
Average the microscopic currents together and you get something very similar.
Average the microscopic currents together and you get something very similar.
Sum these microscopic inwa
This research is from Bezanilla and Correa 1995, Vandenburg and Bezanilla 1991, Correa and Bezanilla 1994
---
## Patch clamping K⁺ channels
@@ -195,7 +198,7 @@ Sum these microscopic inwa
Note:
Now let's examine patch clamp experimental data from K+ channels.
Now let's examine patch clamp experimental data from K+ channels.
---
@@ -217,6 +220,8 @@ Note:
Sustained channel opening in K⁺ channels (outward current).
This research is from Augustine and Bezanilla, Hille 2001; Augustine and Bezanilla 1990; Perozo et al 1991
---
## Measurements of ionic currents flowing through single K⁺ channels
@@ -229,17 +234,19 @@ Sustained channel opening in K⁺ channels (outward current).
</div>
<div style="margin:0 15px"><img src="figs/Neuroscience5e-Fig-04.02-0-2_6ff4997.jpg" height="400px"><figcaption>Neuroscience 5e Fig. 4.2</figcaption></div>
<div style="margin:0 15px"><img src="figs/Neuroscience5e-Fig-04.02-0-2_6ff4997.jpg" height="400px"><figcaption>Neuroscience 5e/6e Fig. 4.2</figcaption></div>
Note:
Sum a bunch of these microscopic channel currents and you get this top curve and which looks very similar to the macroscopic current curve as we’ve seen previously.
Sum a bunch of these microscopic channel currents and you get this top curve and which looks very similar to the macroscopic current curve as we’ve seen previously.
This research is from from Augustine and Bezanilla, Hille 2001; Augustine and Bezanilla 1990; Perozo et al 1991
---
## Functional states of voltage-gated Na⁺ and K⁺ channels
<div><img src="figs/Neuroscience5e-Fig-04.03-0_6eef262.png" width="500px"><figcaption>Neuroscience 5e fig. 4.3</figcaption></div>
<div><img src="figs/Neuroscience5e-Fig-04.03-0_6eef262.png" width="500px"><figcaption>Neuroscience 5e/6e Fig. 4.3</figcaption></div>
Note:
@@ -263,7 +270,7 @@ So the conclusions are…
## Patch clamp method summary video
<div><video height=400px controls src="figs/Animation04-01ThePatchClampMethod.mp4"></video><figcaption>Neuroscience 5e Animation 4.1</figcaption></div>
<div><video height=400px controls src="figs/Animation04-01ThePatchClampMethod_OC.mp4"></video><figcaption>Neuroscience 5e Animation 4.1</figcaption></div>
Note:
@@ -280,7 +287,7 @@ Note:
Note:
Now everything going on in our nervous systems depends on the function of ion channels. And there are lots of them.
Now everything going on in our nervous systems depends on the function of ion channels. And there are lots of them.
---
@@ -291,9 +298,9 @@ Now everything going on in our nervous systems depends on the function of ion ch
Note:
Different classes of gated ion channels.
Different classes of gated ion channels.
voltage gated ion channels, such as we’ve been discussing over the last couple classes.
voltage gated ion channels, such as we’ve been discussing over the last couple classes.
ligand gated channels such as those that bind neurotransmitters, will talk about more later and next class.
@@ -419,7 +426,7 @@ X-ray crystallography
## Structure of the bacterial K⁺ channel
* Bacteria have K⁺ channels that are very similar in structure to mammalian K⁺ channels. Main difference is that they are not gated by voltage
* Bacteria have K⁺ channels that are very similar in structure to mammalian K⁺ channels. Main difference is that they are not gated by voltage
* Could be crystallized in the bacterial membrane
* 3D structure tells us a lot about function
* Roderick Mackinnon Nobel Prize in Chemistry 2003
@@ -436,7 +443,7 @@ eukaryotic
## Structure of the bacterial K⁺ channel
A space-filling model of the KcsA channel, showing the pore. Ions (green balls) tend to occupy three sites in the channel, two in the selectivity filter and one in a pool of water in the center of the channel.
A space-filling model of the KcsA channel, showing the pore. Ions (green balls) tend to occupy three sites in the channel, two in the selectivity filter and one in a pool of water in the center of the channel.
<div><figcaption class="big">red (–) charge; blue (+) charge; yellow hydrophobic</figcaption><img src="figs/image1_eb0191b.jpg" height="300px"><figcaption>Doyle et al, Science 280:69, 1998</figcaption></div>
@@ -472,9 +479,9 @@ Note:
Note:
Simplified model of bacterial K channel, showing you the pore and selectivity filter.
Simplified model of bacterial K channel, showing you the pore and selectivity filter.
helical domains of channel point negative charges towards cavity allowing K ions to become dehydrated and then push through selectivity filter through electrostatic repulsion.
helical domains of channel point negative charges towards cavity allowing K ions to become dehydrated and then push through selectivity filter through electrostatic repulsion.
outside
inside
@@ -514,7 +521,7 @@ Note:
remember water is a polar molecule. Has a net dipole moment of opposing charges in the hydrogen-oxygen bonds.
Larger cations cannot traverse the pore region, smaller cations like Na cannot enter the pore because the walls are just too far apart to stabilize a dehydrated Na ion long enough to pass through.
Larger cations cannot traverse the pore region, smaller cations like Na cannot enter the pore because the walls are just too far apart to stabilize a dehydrated Na ion long enough to pass through.
Na is the most hydrated ion with 4 to 6 water molecules in the first shell. Binds water strongly, making a stable hydration shell and moving together with the cation. Any sodium movement is followed by H2O movement (water retention, excretion).
@@ -531,7 +538,7 @@ K | 0.27 | 0.46
a quote from [http://web-books.com/MoBio/Memory/Channel.htm](http://web-books.com/MoBio/Memory/Channel.htm):
>To pass through the potassium channel, an ion must remove most of its surrounding water molecules, leaving only two - one at the front and another at the back.
>To pass through the potassium channel, an ion must remove most of its surrounding water molecules, leaving only two - one at the front and another at the back.
The selectivity filter of the sodium channel is slightly larger than that of the potassium channel. It may accommodate a Na⁺ ion attached with three water molecules, but not enough for a K⁺ ion attached with three water molecules.
@@ -574,13 +581,13 @@ Now we know from what we’ve learned over the past couple classes that unlike b
[http://web-books.com/MoBio/Memory/Channel.htm :](http://web-books.com/MoBio/Memory/Channel.htm)
>There are many types of potassium channels. The one involved in the generation of action potentials is composed of four subunits, each is homologous to the Shaker protein (Fig. 3.2). The hydrophobicity profile indicates that it contains six hydrophobic segments, designated as S1 - S6. These segments are likely to be the transmembrane domains. Other experimental results suggests that the P-region is lining the channel pore.
>There are many types of potassium channels. The one involved in the generation of action potentials is composed of four subunits, each is homologous to the Shaker protein (Fig. 3.2). The hydrophobicity profile indicates that it contains six hydrophobic segments, designated as S1 - S6. These segments are likely to be the transmembrane domains. Other experimental results suggests that the P-region is lining the channel pore.
<!-- Structure of the subunit of a voltage gated channel protein
<div><img src="figs/sub_struct_99f114c.png" height="100px"><figcaption></figcaption></div>
*N-terminus (amino terminus). Start of a protein or polypeptide. Translation from mRNA occurs from N—>C. Often occurs targeting signals.*
*N-terminus (amino terminus). Start of a protein or polypeptide. Translation from mRNA occurs from N—>C. Often occurs targeting signals.*
*C-terminus (carboxyl terminus), carboxyl group. Often contains retention signals for protein sorting (such as keeping protein in the ER and out of the secretory pathway)* -->
@@ -706,11 +713,11 @@ already learned about tetrodotoxin from puffer fish. blocks voltage gated Na cha
saxitoxin similar (homologue) to ttx, produced by dinoflagellates and possible effects from ‘red tide’ or eating shellfish that have injested these dinoflagellates.
scorpions paralyse prey by injecting alpha-toxins (left panels). Slow inactivation of Na channels, prolonging the AP and messing up information flow in CNS. Beta-toxins in scorpion venom shift the voltage dependence of Na channel activation (right panel), causing Na channels to open at potential much more negative than normal inducing uncontrolled AP firing.
scorpions paralyse prey by injecting alpha-toxins (left panels). Slow inactivation of Na channels, prolonging the AP and messing up information flow in CNS. Beta-toxins in scorpion venom shift the voltage dependence of Na channel activation (right panel), causing Na channels to open at potential much more negative than normal inducing uncontrolled AP firing.
Some alkaloid toxins (batrachotoxin, produced by S. American frogs) do both of these mechanisms.
Similar toxins from plants (aconitine from buttercups, veratridine from lilies) and insecticidal toxins (pyrethrins) produced by chrysanthemums and rhododendrons.
Similar toxins from plants (aconitine from buttercups, veratridine from lilies) and insecticidal toxins (pyrethrins) produced by chrysanthemums and rhododendrons.
dendrotoxin from wasps affects K channels
@@ -728,9 +735,46 @@ charybdotoxin from scorpions K channels
---
## Diseases caused by altered ion channels
## Diseases caused by ion channel mutations
<div style="font-size:0.7em;">
<div style="font-size:0.8em">
<div></div>
* Channelopathies: genetic diseases resulting from mutations in ion channel genes
* e.g. >50 neurological disorders, >40 cardiac disorders ([Kim 2014](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3935107/))
</div>
<div style="float:left; width:300px;font-size:0.6em;">
<div></div>
GEFS: generalized epilepsy with febrile seizures, begins at infancy and continues through puberty. Mapped to two mutations, one on an alpha Na channel subunit and one on a beta subunit. Cause slowing of sodium channel inactivation
Myotonia: muscle contractions
Paralysis: muscle weakness
</div>
<div style="margin:0 20px;"><img src="figs/Neuroscience5e-Box-04D-2R_d27c5d4.jpg" height="300px"><figcaption>Neuroscience 5e Box 4D;
see also Neuroscience 6e 'Clinical applications' p. 75-77</figcaption></div>
Note:
More than 20 different inhereited diseases from from mutations in Na channels alone. Cystic fibrosis results from chloride channel dysfunction (and altered fluid movements, chloride gradients often used for cell volume, fluid movements).
ataxia: greek for ‘without order’ or ‘incoordination’. Movement coordination problems.
paralysis: muscle weakness
myotonia: muscle contraction
<!--
## Diseases caused by altered ion channels
<div style="float:left; width:300px;font-size:0.6em;">
<div></div>
EA1: episodic ataxia type 1 (abnormal limb movements and severe ataxia)
@@ -739,25 +783,13 @@ BFNC: benign familial neonatal convulsion. Frequent brief seizures starting in f
</div>
<figure><img src="figs/Neuroscience5e-Box-04D-3R_2e54724.jpg" height="300px"><figcaption>Neuroscience 5e Box 4D</figcaption></figure>
Note:
ataxia: greek for ‘without order’ or ‘incoordination’. Movement coordination problems.
paralysis: muscle weakness
myotonia: muscle contraction
--
<div style="margin:0 20px">
<img src="figs/Neuroscience5e-Box-04D-3R_2e54724.jpg" height="300px">
<figcaption>Neuroscience 5e Box 4D;
see also Neuroscience 6e 'Clinical applications' p. 75-77</figcaption>
</div>
## Diseases caused by altered ion channels
<div style="font-size:0.7em;">
<div></div>
@@ -773,56 +805,32 @@ Paralysis: muscle weakness
<figure><img src="figs/Neuroscience5e-Box-04D-1R_d538656.jpg" height="300px"><figcaption>Neuroscience 5e Box 4D</figcaption></figure>
Note:
nystagmus: involuntary eye movements (dancing eyes)
strabismus: eyes not directed towards same fixation point, disruption of binocular vision, depth perception, resulting in amblyopia when present in children
amblyopia: greek for ‘blunt vision’, decr vision through an eye because of a developmental pathophysioloy of the brain (e.g. visual cortex), 1-5% of population
--
## Diseases caused by altered ion channels
<div style="font-size:0.7em;">
<div></div>
GEFS: generalized epilepsy with febrile seizures, begins at infancy and continues through puberty. Mapped to two mutations, one on an alpha Na channel subunit and one on a beta subunit. Cause slowing of sodium channel inactivation
Myotonia: muscle contractions
Paralysis: muscle weakness
</div>
<figure><img src="figs/Neuroscience5e-Box-04D-2R_d27c5d4.jpg" height="300px"><figcaption>Neuroscience 5e Box 4D</figcaption></figure>
Note:
-->
--
## Epilepsy can result from mutated Na⁺ channels
<figure><img src="figs/Neuroscience5e-Box-04D-5R_d4ec056.jpg" height="300px"><figcaption>Neuroscience 5e Box 4D</figcaption></figure>
<figure><img src="figs/Neuroscience5e-Box-04D-5R_d4ec056.jpg" height="400px"><figcaption>Neuroscience 5e Box 4D;
see also Neuroscience 6e 'Clinical applications' p. 75-77</figcaption></figure>
Note:
You can see the the slower inactivation kinetics in this figure here in patch clamp recordings from normal and a number of different Na channel mutants. This slowing of Na inactivation is just enough to mess up spike patterns in single neurons and elicit hyperexcitability that results in seizures in networks of connected neurons.
You can see the the slower inactivation kinetics in this figure here in patch clamp recordings from normal and a number of different Na channel mutants. This slowing of Na inactivation is just enough to mess up spike patterns in single neurons and elicit hyperexcitability that results in seizures in networks of connected neurons.
GEFS: generalized epilepsy with febrile seizures
<!-- ## Impaired inactivation of Na⁺ channels underlie hyperkalemic periodic paralysis disease
* Normally Na⁺ channels open and close rapidly due to inactivation.
* Normally Na⁺ channels open and close rapidly due to inactivation.
* Na⁺ channels from hyper periodic paralysis close more slowly and reopen.
* Mutant myotubes (Met 1592 Val)
* Mutant myotubes (Met 1592 Val)
<div><img src="figs/image7_82d08af.png" height="100px"><figcaption>Cannon et al, Neuron 1991</figcaption></div>
@@ -841,17 +849,25 @@ GEFS: generalized epilepsy with febrile seizures
## Ion transporters
<figure><img src="figs/Neuroscience5e-Fig-04.09-0_996247d.jpg" height="300px"><figcaption>Neuroscience 5e Fig. 4.9</figcaption></figure>
<figure><img src="figs/Neuroscience5e-Fig-04.09-0_996247d.jpg" height="300px"><figcaption>Neuroscience 5e Fig. 4.9, 6e Fig. 4.13</figcaption></figure>
Note:
Lastly let's remind ourselves of the importance of ion transporters in maintaining the concentration gradients across the nerve cell membrane. We've previously discussed the active transporter the Na/K pump that is crucial for maintaining Na/K gradients but there are others that maintain gradients for other physiologically relevant ions like Cl, Ca.
Lastly let's remind ourselves of the importance of ion transporters in maintaining the concentration gradients across the nerve cell membrane. We've previously discussed the active transporter the Na/K pump that is crucial for maintaining Na/K gradients but there are others that maintain gradients for other physiologically relevant ions like Cl, Ca.
Remember these transporters are all very slow compared to ion channels, **requiring several milliseconds to move a few ions** compared to **thousands of ions per second** conducted across the membrane for an ion channel.
Crystal structure for Na/K channel with either K bound in the central pore or Na was just solved in 2009 and 2013 respectively (Shinoda et al, Nature 2009) Nyblom et al. Science 2013)
<!-- ## Na⁺/K⁺ pump video
<div><video height=400px controls src="figs/Animation04-02TheSodiumPotassiumPump.mp4"></video><figcaption>Neuroscience 5e Animation 4.2</figcaption></div> -->
<div><video height=400px controls src="figs/Animation04-02TheSodiumPotassiumPump_OC.mp4"></video><figcaption>Neuroscience 5e Animation 4.2</figcaption></div> -->
Ouabain, plant 'arrow' poison traditionally from africa from the Acokanthera schimperi and Strophanthus gratus plants. Binds to the Na+/K+ pump. Cardiac dysfunction ensues.
Used together with
Radioactive Na efflux measurements and radioactive K influx measurements used with ATP synthesis inhibitors (e.g dinitrophenol) to help demonstrate that an active Na/K pump is responsible for producing ion conentraiton gradients in squid axon (Hodgkin and Keynes 1955).