Exam 2 Powerpoint 10 Flashcards
H and H experiments predicted
existence of channels
Predictions (4)
Channels must allow ions to move across the membrane at a high rate
Channels make use of electrochemical gradients
Discriminating –specific for specific ions
Voltage sensitive; inactivating in some cases
Technical impediment H and H faced
Voltage clamp allowed recording of
aggregate currents (thousands of channels): needed a new
method
Patch Clamping was invented in
1976
Patch clamping was made to
record activity of a single ion channel or whole cell currents
Patch clamping forms a
high resistance seal between cell membrane and glass wall of
the micropipette (apply suction)
Patch clamp recording allows
Allows recording from one ion channel
No ions escape b/c it is a very tight seal
Low level of background noise
Can alter the potential across the patch simply by applying
voltage to the pipette
https://www.youtube.com/watch?v=mVbkSD5FHOw
https://www.youtube.com/watch?v=TUoCQTwewVo
Sodium currents Recorded in the
presence of _____ which blocks
__________ channels
Cs+
Potassium
Macroscopic recordings are from
multiple channels
Microscopic recordings are from
1 channel
Depolarization increases the probability of
channel opening
Potassium currents
in in presence of _____
which blocks __________________
TTX
sodium
channels
___genes are associated with K channels
78
____genes are associated with Na channels
29
More than _____ genes encode for channels. Can vary due to RNA editing and splice variants.
230
Why is it difficult to study specifics in vivo by patching onto neurons
Hard to tell individual channels apart (lots of noise)
Challenging in telling different types of neurons apart
Splice variants
Pharmacological tools are not always great, although not always available
The molecular structures of ion channels as individual proteins were determined after the cloning of genes encoding specific ion channels, as the revolution in
molecular biology spread to neuroscience in the 19__s.
80s
Voltage-gated ____ channel proteins were first purifed from the electric eel Electrophorus electricus, whose electric organ is densely packed with ___ channels
Na+
Xenopus System
1980s first demonstration that they can express ion channels
Harvest eggs from females
1 mm diameter
Once you inject mRNA (you synthesize in vitro), makes a lot of protein
Powerful—combine physiology and molecular methods
Xenopus Oocytes
Immature eggs from the clawed African frog (Xenopus laevis) used as
expression machines
Channel genes can come from any organism (the beauty of a common
central dogma)
How do we study channels
Use of well defined systems
(heterologous systems)
-Cell lines (cells in a dish)
-Frog oocytes
Genetic mutations in model
organisms
-Mice
-Fruit flies
Natural toxins for sodium channels
TTX (puffer fish)- paralyze
Saxitoxin (red tide dinoflagellates)-
similar to TTX
Scorpion toxins-alpha toxins
(paralysis, arrhythmias)-slow
inactivation of VGSC
Natural toxins for potassium channels
x- TEA, cesium
x- Dendrotoxin (from wasps, snakes)
(causes convulsions)
- Block repolarization—prolong
action potentials
x- Apamin (bee)
Major channel structure feature
Plasma membrane
Channels insert
between membrane spanning structures, pores
Selectivity filter:
Size of cation is a big factor
Multiple subunits:
Repeating motifs within
these subunits
Voltage gated channels have specific functional states:
Open
Inactivating
Inactivated
Closed
States of a Voltage gated Na+ channel
Closed
open
inactivating
inactivated
closed
States of a Voltage gated K+ channel
Closed
Closed
Open
Open
Closed
Voltage sensing
Depolarization changes the structure
How voltage sensing result in channel opening
X-ray Crystallography studies: structure
Mutational analysis: understand the important amino acids
Combine mutational analysis with electrophysiology
Positively charged amino acids in protein channels are
voltage sensors
Na+ and Ca2+ channels:
1 large subunit: 4 repeat motifs with 6 membrane
spanning regions each (total: 24 transmembrane segments)
K+ channels:
4 subunits come together to make a functional channel
Cl- channels structurally distinct:
2 separate pores made by two sets of 2 identical subunits
Auxiliary subunits such as beta, gamma are
Often important for channel functionality, such as inactivating
