Lecture 10 - membrane potential and ion transport 2 Flashcards
what do ion channels have?
many TM domains
what does the patch clamp measurements reveal?
the activities of single channels
what are the patch clamp measurements?
Excised patches allow:
measurement of currents through individual channel proteins
control over ion concentrations at both sides of the membrane
control over membrane potential (voltage ‘clamp’)
Current plotted against clamped voltage = I/V curve
conductance of the channel: steepness
reversal potential: voltage where I = 0 (+15mV in the picture)
compare reversal potential with equilibrium potentials of the ions present to obtain relative selectivity of the channel for the different ions
open probability: frequency of opening
gating: change of open probability
e.g. by voltage (inward rectifying, outward rectifying…) or ligands
what are channel properties?
Patch-clamp experiments revealed that
-Channels can be highly selective for certain ions
- Channels have a very high transport rate >108 ions per second (conductance)
- Channels open and close (‘gating’)
- Channels sometimes inactivate after opening
what is the P-type ATPase cycle?
Binding of ATP and subsequent phosphorylation of a conserved Aspartate residue re-arranges
A,P,N domains and subsequently TM domain so that two Ca2+ ions are released into SR.
De-phosphorylation of the Asp residue converts the protein back into its original state.
what are p-type ATPases?
e.g. Ca2+ ATPase (SERCA): Pumps Ca2+ from the cytoplasm into sarcoplasmic reticulum (SR) of muscle cells
Muscle contraction is triggered by high Ca2+ concentration in cytoplasm = Ca2+ signal
The signal is caused by release of Ca2+ from the SR.
During muscle relaxation the Ca2+ is pumped back from the cytoplasm into the SR .
Concentration difference to be achieved in SR/Cytoplasm = 1.5 mM/0.1 mM = 15,000 fold
Needs input of energy in the form of ATP.
what is the acetylcholine receptor?
Acetylcholine receptor is gated by the ligand acetylcholine.
It is a non-selective cation channel
permeable to both K and Na.
Opening of the channel results in a small depolarisation of the membrane potential
(by ca. 20mV).
This small initial change in membrane potential triggers an action potential.
Opening of the channel is achieved
by rotating the M2 TM helix by 15º, which allows the inner ring to expand
what is the potassium channel pore?
3Å diameter is wide enough for K+ and Na+ ions
– why then is the channel K+ selective?
To get from wide (blue) into the narrow (yellow) part of the pore ion has to remove the hydration shell
what is the high rate of transport?
Tight binding of ions in the selectivity filter should slow them down.
Why does it not?
There are 4 binding sites inside the selectivity filter
Ions push one another one site up due to the electrostatic repulsion
what are the Methods for structure-function analysis?
Heterologous expression of membrane proteins in Xenopus oocytes
- Clone channel gene in E.coli.
- Carry out in-vitro transcription
- Inject RNA into oocytes
- Measure ion currents
what is the Channel inactivation: Ball-and-Chain Model?
The Ball-and-Chain Model of channel inactivation is a classic explanation for how voltage-gated ion channels (like sodium channels) stop conducting ions shortly after opening.
In this model, after the channel opens, a part of the protein (the “ball”) connected by a flexible linker (the “chain”) swings around and blocks the inner mouth of the channel from the cytoplasmic side. This inactivates the channel, preventing further ion flow, even though the activation gate remains open.
When the membrane repolarizes, the ball unplugs, and the channel returns to a resting state, ready to be activated again.
