32 KiB
Ion channels underlie action potentials
- Predictions about the nature of ion channels from Hodgkin and Huxley:
- Because conductances are large, channels must be able to pass ions at high rate.
- Channels must be gated by the membrane potential.
- Different channels for Na+ and K+.
- Problem– Voltage clamping cannot look at individual channels…its measuring the aggregate current flowing through a whole bunch of channels at once. What does an individual channel look like? How does it work?
- Solution– Patch Clamping
2016-01-19 17:18:07
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Today we will take a closer look at how ion channels are able to exhibit their remarkable properties and enable action potentials and all forms of electrical signaling in the nervous system.
Now we know from our previous classes covering the work by HH, that there are some predictions we make concerning the nature of ion channels:
Question
- During the rising phase of the action potential:
- a. All sodium channels are closed.
- b. Some of the sodium channels are closed
- c. All potassium channels are open.
- d. All sodium channels are open.
- e. The membrane potential is hyperpolarizing.
During the rising phase of the action potential:
a. All sodium channels are closed.
b. Some of the sodium channels are closed
c. All potassium channels are open.
d. All sodium channels are open.
e. The membrane potential is hyperpolarizing.
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So a quick question
The Nobel Prize in Physiology or Medicine (1991)
“for their discoveries concerning the the function of single ion channels in cells”
Erwin Neher
Bert Sakmann
http://nobelprize.org/nobel_prizes/medicine/laureates/1991/press.html
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Title Text
- What ions are permeable at rest?
- What ions are permeable at peak?
- What ions are permeable during overshoot?
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How would the shape of the action potential change if the extracellular Na concentration was lowered, what if the K+ was raised
Patch clamp method:
Neher and Sakmann
cell-attached recording
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Patch clamp
- Allows one to look at currents flowing through a single channel.
- Pipette with small opening makes a tight seal with the membrane.
- Currents are amplified and measured
- Can be adapted to do whole cell recordings, inside out recordings or outside out recordings.
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The patch clamp method
- Can measures ion flow through a single channel
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The patch clamp method
Pipette is continuous with cytoplasm. Can measure potentials and currents from the entire cell and also can introduce things into the cytoplasm
Makes it easy to introduce things to the cytoplasmic side of the channel
Makes it easy to introduce
things to the extracellular
side of the channel
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Patch clamping Na+ channels
- Block K+ channels with Cs+ or with tetraethylammonium (TEA).
- Brief depolarizations cause small inward currents that disappear right away.
- Each inward current is the opening of one Na+ channel.
- About 1-2 pA of current == thousands of ions/ms
- Stochastic opening, biased at the beginning of a pulse.
- Probability of opening varies with membrane potential.
- If you remove Na+ from medium, do not see currents.
- Tetrodotoxin (TTX) blocks currents.
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Measurements of ionic currents flowing through single Na+ channels
Small inward currents
Open at beginning of pulse
Close quickly
Neuroscience5e Fig. 4.1
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Patch a piece of membrane and block K currents. Do a bunch of short recordings while clamping the membrane at depolarized potential. e.g. here is 7 trials. Notice the amplitude is discrete— it is unitary. If you were recording from lots of…
Transient channel opening in Na+ channels (inward current).
Measurements of ionic currents flowing through single Na+ channels
Neuroscience5e Fig. 4.1
Summed current from many single channels looks like macroscopic currents seen in voltage clamping
Probability of opening increases as a function of membrane potential
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You’d expect this macroscopic current—
Average the microscopic currents together and you get something very similar.
Sum these microscopic inwa
Patch clamping K+ channels
- Add tetrodotoxin (TTX) to block Na+ channels
- Depolarization pulses cause outward currents.
- Once a channel opens (usually with a delay) it remains open for the duration of the pulse.
- The probability of channel opening depends on the membrane potential.
- Sensitive to TEA.
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Measurements of ionic currents flowing through single K+ channels
Early delay in opening
Once open stay open
Neuroscience5e Fig. 4.2
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Sustained channel opening in K+ channels (outward current).
Measurements of ionic currents flowing through single K+ channels
Summed current from many single channels looks like macroscopic currents seen in voltage clamping
Probability of opening increases as a function of membrane potential
Neuroscience5e Fig. 4.2
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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.
Functional states of voltage-gated Na+ and K+ channels
Neuroscience5e Fig. 4.3
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Shown here is a model of the functional states for these channels. Notice a few states for Na vs two for K.
Conclusions from patch clamp experiments
- Allowed the direct observation of ionic currents flowing through single ion channels.
- Both Na+ and K+ channels are voltage gated.
- Thus there must be a voltage sensor in these channels.
- Depolarization inactivates Na+ channels but not K+ channels.
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So the conclusions are…
Title Text
http://courses.pbsci.ucsc.edu/mcdb/bio125/Animation04-01ThePatchClampMethod.mov
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Many genes encode ion channels
- There are hundreds of genes encoding ion channels (e.g. about 100 K+ channel genes).
- They have common properties (similarities in amino acid sequence and protein topology).
- They also have variations (differences in ion selectivity, how they are gated, inactivation mechanisms).
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Now everything going on in our nervous systems depends on the function of ion channels. And there are lots of them.
Different ways to gate ion channels
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Different classes of gated ion channels.
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.
others are ligand gated channels sensitive to chemical signals arising in the cytoplasm of neurons such second messengers like Ca2+, cyclic nucleotide cAMP and cGMP.
Even within a family of channelsthere is huge variation
- Voltage gated– Na+, K+, Cl-, and Ca2+ channels.
- Approximately 10 different genes for Na+ channels, 16 Ca2+, 3–5 Cl- and 100 K+ channels.
- Different genes may give rise to channels with different properties– e.g. different inactivation times, probability of opening at a given voltages, gating mechanisms.
- Can also be multiple splice variants of the same gene.
- Creates huge diversity of channels.
- How to characterize all these channels?
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Lets say you have a gene for a channel, how do you determine its properties
- Need an experimental system where you can express gene of interest functionally and away from other channels.
- Xenopus oocytes have been a historical way to do this.
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frog germ cells
Xenopus oocytes
- Large (1 mm in diameter) cell that contains lots of protein synthesis machinery.
- Can inject RNA into it and it will express protein encoded by RNA.
- Works great for ion channels, can voltage clamp and determine properties of a given channel.
- Can make specific mutations in genes and see what happens to function of protein.
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Xenopus oocytes for ion channel physiology studies
Channel gene (DNA)
Transcribe in vitro
mRNA
Oocyte makes protein
Whole cell record or
patch clamp channel
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Xenopus oocytes for ion channel physiology studies
Ion channel mRNA
Oocyte makes protein
Whole cell record or
patch clamp channel
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Xenopus oocytes for ion channel physiology studies
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shows voltage clamp experiment results after expression of a K channel in an oocyte.
Diverse properties of K+ channels
Neuroscience5e Fig. 4.5
Similar to action potential
K+ channels
Inactivates like Na+ channels
Same conductance vs voltage profiles, different inactivation properties.
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-
Kv2.1 show little inactivation and are closely related to the delayed rectifier K channels involved in AP repolarization
-
Kv4.1 channels inactivate during a depolarization.
HERG channels inactivate so rapidly that current flows only when inactivation is rapidly removed at end of a depolarization
inward rectifier K channels allow more K current to flow at hyperpolarized potentials than at depolarized potentials
Ca activated K channels open in response to intracellular Ca ions
2-P K channels usually respond to chemical signals rather than changes in membrane potential. These are primarily responsible for the resting membrane potential of neurons. e.g. TASK channels can by regulated by extracellular pH
Diverse properties of K+ channels
More current flows when
hyperpolarization conditions
More current flows if Ca2+ is
added intracellularly
pH sensitive channel
Neuroscience5e Fig. 4.5
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-
Kv2.1 show little inactivation and are closely related to the delayed rectifier K channels involved in AP repolarization
-
Kv4.1 channels inactivate during a depolarization.
HERG channels inactivate so rapidly that current flows only when inactivation is rapidly removed at end of a depolarization
inward rectifier K channels allow more K current to flow at hyperpolarized potentials than at depolarized potentials
Ca activated K channels open in response to intracellular Ca ions
2-P K channels usually respond to chemical signals rather than changes in membrane potential. These are primarily responsible for the resting membrane potential of neurons. e.g. TASK channels can by regulated by extracellular pH
Molecular structures of ion channels
- Multiple membrane spanning domains
- K+ channels 4 subunits come together, (each with 6 transmembrane helices).
- Na+ channels 1 protein with 24 transmembrane helices
- Center has an opening that makes a pore for the ion to flow through
- Contains selectivity filter
- Voltage-gated ion channels also contain a voltage sensitive transmembrane domain
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We’ve learned from biophysical structure studies that in general ion channels have 24 transmembrane peptide domains with…
We can also guess a few characteristics of their structure from the classic voltage clamp and patch clamp studies we’ve discussed over the past couple classes…
from wikipedia:
X-ray crystallography is a tool used for identifying the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.
Molecular structures of ion channels
- Voltage-gated cation channels consist of four subunits, each of which has 6 transmembrane segments and a pore loop. In sodium and calcium channels, the four subunits are part of the same molecule. In potassium channels, they are different molecules.
The resulting channel has four-fold symmetry
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Molecular structures of ion channel proteins
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-
Kv2.1 show little inactivation and are closely related to the delayed rectifier K channels involved in AP repolarization
-
Kv4.1 channels inactivate during a depolarization.
HERG channels inactivate so rapidly that current flows only when inactivation is rapidly removed at end of a depolarization
- human Ether-à-go-go-Related Gene), best known for its contribution to the electrical activity of the heart that coordinates the heart's beating, mediates the repolarizing IKr current in the cardiac action potential).
inward rectifier K channels allow more K current to flow at hyperpolarized potentials than at depolarized potentials
Ca activated K channels open in response to intracellular Ca ions
2-P K channels usually respond to chemical signals rather than changes in membrane potential. These are primarily responsible for the resting membrane potential of neurons. e.g. TASK channels can by regulated by extracellular pH
Channel selectivity
can get through a Na channel
Can’t
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Potassium channel with four subunits
Crude understanding of structure– 4 subunits come together to
make a pore.
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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.
- Could be crystallized in the bacterial membrane.
- 3D structure tells us a lot about function.
- Roderick Mackinnon Nobel Prize in Chemistry 2003
“for structural and mechanistic studies of ion channels”
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prokaryotic
eukaryotic
Structure of the bacterial K+ channel
3D structure of bacterial K channel. Yellow is the K channel,
white are phospholipids, purple Na, green K.
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Structure of a bacterial K+ channel determined by crystallography
helps dehydrate
K+ ions
inside
outside
Neuroscience5e Fig. 4.7
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Simplified model of bacterial K channel, showing you the pore and selectivity filter.
helical domains of channel point negative charges towards cavity allwing K ions to become dehydrated and then push through selectivity filter through electrostatic repulsion.
Structure of the bacterial K+ channel
Each subunit has 2
transmembrane domains, 4 subunits make a channel
Neuroscience5e Fig. 4.7
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(Doyle et al, Science 280:69, 1998)
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.
red – charge; blue + charge; yellow hydrophobic
(Doyle et al, Science 280:69, 1998)
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(Doyle et al, Science 280:69, 1998)
Selectivity filter of the K+ channel
Up to 4-6 water molecules form hydration shells around both Na+ and K+ ions
Ions move with their hydration shells
To pass through the potassium channel, an ion must remove most of its surrounding water molecules (dehydrated)
K+ is dehydrated by the K+ channel selectivity filter (leaving just two water molecules– one at front and one at back)
Na+ has a more stable water shell, binding H2O more strongly and thus has a larger effective diameter— would require more dehydration energy than K channel pore region can provide.
H2O
K+
K+
dehydrate, move through filter
filter
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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).
Potassium ion is larger, having 8 more electrons shielding positively charged nucleus, thus K+ makes transient associations with water rather than a discrete hydration layer. Helps explain higher permeability across cell membrane for K+.
ion | ion diameter (nm) free | ion diameter hydrated
--- | ---------------------- | -------------------
Na | 0.19 | 0.52
K | 0.27 | 0.46
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.
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.
In the sodium channel, the Na+ ion is more permeable than the K+ ion. This is because the selectivity filter of the sodium channel is slightly larger than that of the potassium channel. It is large enough to accommodate a Na+ ion attached with three water molecules, but not enough for a K+ ion attached with three water molecules. Therefore, to pass through the sodium channel, the Na+ ion needs to remove only three, but the K+ ion has to remove four, water molecules from its first hydration shell. The required dehydration energy for the K+ ion is greater than the Na+ ion.
In calcium channels, the permeability of monovalent cations (Na+ and K+) is about three orders of magnitude smaller than the Ca2+ permeability. This ion selectivity does not seem to involve hydration, because Ca2+ is more heavily hydrated than Na+, and the unhydrated diameters of Ca2+ and Na+ are almost identical. Then, how could calcium channels select Ca2+ over Na+?
Although the permeability of monovalent cations in the calcium channel is quite small at normal ionic concentrations, large monovalent cationic current can be observed in the absence of Ca2+ and other divalent cations. This suggests that the calcium channel is basically permeable to both divalent and monovalent cations, but the selectivity arises from competition between ions. The calcium channel may contain a negatively charged binding site to facilitate ion conduction. The monovalent cations simply cannot compete with Ca2+ for this binding site. This idea has been confirmed experimentally. In the calcium channel, if a negatively charged glutamate residue in the pore-lining region is mutated into a positively charged lysine, the calcium channel becomes more permeable to Na+ than Ba2+
This explains the selectivity but not the voltage sensor
Atomic masses
1H < 2He
3Li < Be < B < 6C < N < 8O < 9F < 10Ne
11Na < 12Mg < Al < 14Si < P < S < 17Cl < 18Ar
19K < 20Ca
37Rb < 38Sr
55Cs < 56Ba
Structure of the subunit of a voltage gated
channel protein
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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)
Structure of a mammalian voltage-gated K+
Neuroscience5e Fig. 4.8
top view
side view
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Now we know from what we’ve learned over the past couple classes that neurons have K+ channels that are gated by voltage
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.
Structure of a mammalian voltage-gated K+
Neuroscience5e Fig. 4.8
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Topology of the principal subunits of voltage-gated Na+ channels
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Yellow are voltage sensing tm domains
Topology of the principal subunits of voltage-gated Ca2+ channels
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Yellow are voltage sensing tm domains
Topology of the principal subunits of voltage-gated K+ channels
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K channels are more diverse
Yellow are voltage sensing tm domains
How do Na+ channels inactivate?
- Contains an activation gate that binds to the channel in the intracellular region and blocks the channel
- Activation gate changes conformation (closes/swings shut) to block channel only during the channel’s open state
- Therefore, at resting Vm channel is closed and activation gate is open.
- After depolarization, the channel opens and Na+ ions go through. After a little bit of time (~ 1 ms) the activation gate swings shut to block channel.
http://www.nature.com/nature/journal/v475/n7356/full/nature10238.html
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The theory is that the inactivation gate “swings” shut, turning off the channel
The physical structure of voltage gated Na channels has only recently begun to be solved, with the results so far fitting the models for Na channel opening and inactivation.
Sodium channel inactivation cycle
Lodish, Mol Cell Bio
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Figure 21-13 Lodish 4th edition OR Figure 7-33 Lodish 5th edition. Structure and function of the voltage-gated Na+ channel.
http://www.amazon.com/Molecular-Cell-Biology-Lodish/dp/0716776014
Sodium channel inactivation
The theory is
that the inactivation gate
“swings” shut, turning off
the channel
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The theory is that the inactivation gate “swings” shut, turning off the channel
Toxins that poison ion channels
prolongs Na+ currents
by messing up channel inactivation
AP profile reflects
the shift in Na+
conductance.
http://www.nature.com/news/rodent-immune-to-scorpion-venom-1.14014
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already learned about tetrodotoxin from puffer fish. blocks voltage gated Na channels underlying the AP
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.
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.
dendrotoxin from wasps affects K channels
apamin from bees K channels
charybdotoxin from scorpions K channels
Toxin binding sites
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Blue diamonds- persistent activation
Picture from sigma catalog of blockers
Diseases caused by altered ion channels
EA1: episodic ataxia type 1 (abnormal limb movements and severe ataxia)
BFNC: benign familial neonatal convulsion. Frequent brief seizures starting in first postnatal week then disappearing in a few months. Mutation mapped to two K+ channel genes
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ataxia: greek for ‘without order’ or ‘incoordination’. Movement coordination problems.
paralysis: muscle weakness
myotonia: muscle contraction
Diseases caused by altered ion channels
FHM: familial hemiplegic migraine; CSNB: congenital stationary night blindness, rod photoreceptors nonfunctional (resulting in decreased acuity, myopia, nystagmus, strabismus); EA2: episodic ataxia type 2 (abnormal limb movements and severe ataxia); Paralysis: muscle weakness
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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
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
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Diseases caused by altered ion channels
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Epilepsy can result from mutated Na+ channels
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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.
- Na+ channels from hyper periodic paralysis close more slowly and reopen.
- Mutant myotubes (Met 1592 Val)
Cannon et al, Neuron 1991
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autosomal dominant disorder characterized by episodic weakness lasting minutes to days in association with a mild elevation in serum K+
whole-cell currents in HPP muscle have demonstrated a persistent, tetrodotoxin-sensitive Na+ current
linkage analysis that the Na+ channel alpha subunit gene may contain the HPP mutation
patch-clamp recordings from cultured HPP myotubes and found a defect in the normal voltage-dependent inactivation of Na+ channels
Muscle fibers generally form from the fusion of myoblasts into multi-nucleated fibers called myotubes
Channelopathies
- Diseases can be linked to defective ion channels
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Ion transporters
Neuroscience5e Fig. 4.9
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Title Text
http://courses.pbsci.ucsc.edu/mcdb/bio125/Animation04-02TheSodiumPotassiumPump.mov
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