* 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...it's measuring the aggregate current flowing through a whole bunch of channels at once. What does an individual channel look like? How does it work?
Today we will take a closer look at the nature of **ion channels** and how they are able to exhibit their remarkable properties that enable action potentials and all forms of electrical signaling in the nervous system.
* 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
Note:
Remember voltage-clamp recordings that we've been talking about before. Patch-clamping is an adaptation of the voltage-clamp method that allows you to assess the currents flowing across very small patches of lipid membrane. So if you have one or few ion channels in that small patch of cell membrane you can study the microscopic functional properties of those individual channels.
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## The patch clamp method
Can measure ion flow through a single channel.
<figure><img src="figs/Neuroscience5e-Box-04A-1R_copy_b363c83.jpg" height="300px"><figcaption>Neuroscience 5e Box 4A</figcaption></figure>
Note:
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## The patch clamp method
<div style="height:300px"><figcaption>Neuroscience 5e Box 4A</figcaption><img src="figs/Neuroscience5e-Box-04A-2R_58077da.jpg" height="200px"><figcaption class="big">
Can measure potentials and currents
from entire cell and introduce
things into the cytoplasm
</figcaption></div>
<div style="margin:0 25px; height:300px"><figcaption>Neuroscience 5e Box 4A</figcaption><img src="figs/Neuroscience5e-Box-04A-3R_8f113be.jpg" height="200px"><figcaption class="big">
Makes it easy to introduce things to
the cytoplasmic side of the channel
</figcaption></div>
<div><figcaption>Neuroscience 5e Box 4A</figcaption><img src="figs/Neuroscience5e-Box-04A-4R_1677b63.jpg" height="200px"><figcaption class="big">
Makes it easy to introduce things to the extracellular side of the channel
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 these single channels simultaneously or added together all the recordings from one channel you'd -->
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
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.
* 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
from [channelpedia](http://channelpedia.epfl.ch/ionchannels/9):
>Kv2.1 is widely expressed in brain and composes the majority of delayed rectifier K+ current in pyramidal neurons in cortex and hippocampus and is also widely expressed in interneurons. Dynamic modulation of Kv2.1 localization and function by a mechanism involving activity dependent Kv2.1 dephosphorylation dramatically impacts intrinsic excitability of neurons.
- Kv4.1 channels inactivate during a depolarization.
>a voltage-activated A-type potassium ion channel and is prominent in the repolarization phase of the action potential. This gene is expressed at moderate levels in all tissues analyzed, with lower levels in skeletal muscle.
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
<figure><img src="figs/Neuroscience5e-Fig-04.05-3R_6f6bb99.jpg" height="400px"><figcaption>Neuroscience 5e Fig. 4.5</figcaption></figure>
- 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
* 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
Note:
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…
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.
<figure><figcaption class="big">Each subunit has 2 transmembrane domains, 4 subunits make a channel</figcaption><img src="figs/Neuroscience5e-Fig-04.07-2R_5838376.jpg" height="400px"><figcaption>Neuroscience 5e Fig. 4.7</figcaption></figure>
Note:
(Doyle et al, Science 280:69, 1998)
<!-- ## Structure of the bacterial K⁺ channel
3D structure of bacterial K channel. Yellow is the K channel, white are phospholipids, purple Na, green K.
helical domains of channel point negative charges towards cavity allowing K ions to become dehydrated and then push through selectivity filter through electrostatic repulsion.
* 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 H<sub>2</sub>O more strongly and thus has a larger effective diameter— would require more dehydration energy than K channel pore region can provide
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⁺.
>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+
>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.
*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)* -->
4–8 positively-charged amino acids in the S4 domain. Experiences force in a transmembrane electric field. Is the electric-field sensor for voltage-dependent gating.
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
* Therefore, at resting V<sub>m</sub> 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
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.
<figure><figcaption class="big">prolongs Na⁺ currents by messing up channel inactivation</figcaption><img src="figs/Neuroscience5e-Box-04B-1Ra_1ce75c5.jpg" height="200px"><figcaption>Neuroscience 5e Box 4B</figcaption></figure>
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.
Similar toxins from plants (aconitine from buttercups, veratridine from lilies) and insecticidal toxins (pyrethrins) produced by chrysanthemums and rhododendrons.
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.
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
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
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.
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)
Ouabain, plant 'arrow' poison traditionally from africa from the Acokanthera schimperi and Strophanthus gratus plants. Binds to the Na+/K+ pump. Cardiac dysfunction ensues.
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).
