Experiments 1 Flashcards
How does the voltage clamp work?
The voltage clamp amplifier allows the researcher to select a particular membrane potential (the “command” or “clamp” potential) and to hold (= clamp) the membrane of a cell at that potential. If there are ion movements across the membrane that would change the membrane potential, the voltage clamp amplifier detects any movement of the membrane potential away from the “command” potential and generates a current that will bring back the membrane potential to its clamped value (feedback). By measuring the current produced by the device to keep the membrane potential constant under various experimental conditions, researchers can measure quantitatively the ion currents produced by the neurone under those conditions
Outline the aims and methods for Hodgin & Huxley’s experiments in the 1950’s
Aim: to provide a quantitative description of the current flow contributed by each ion and how that current flow leads to the final shape and amplitude of an action potential.
Method: Hodgkin and Huxley used the voltage clamp technique to study ion flux in squid giant axons. Their recordings demonstrated the existence of two types of voltage-gated currents. They used 3 different methods to separate current components:
By modifying the concentration gradient of one of the critical ions (i.e. Na+);
By substituting one of the critical ions (i.e. Na+) with an impermeant one;
By selective pharmacological blockade of the different voltage-sensitive channels (i.e. TTX for Na+ channels, TEA for K+ channels)
Which experiments formed the basis of ion channel theory?
Ionic basis of AP -> The basis for ion channel theory came from classic experiments with Cole, Curtis, Hodgkin, Huxley and Katz using voltage clamp where differences in permeability to different ions was first observed. Idea of channels from Hodgkin + Keynes which led to Armstrong + hill’s working hypothesis of ion channels.
Outline the ion channel theory
Initial inward current caused by Na+ flux -> remaining current when choline used is outward K+ flux
- > systematic measurement of Na+ and K+ conductance over a series of different voltages
- > conductance = ratio of current that passes through membrane + driving force -> different between membrane potential and equilibrium potential
Who invented the patch clamp and how does it work?
Neher & Sakmann 1976
-> fire polished glass micropipette 3-5 um in diameter to electrically isolate membrane -> pipette is filled with ringer solution and agonist - interior is connected to voltage circuit and recorder + conventional 2 micro electrode voltage clamp for interior of muscle fibre -> first time measure single channel conductance with high resolution using amplitude histograms - looking at ACh + frog nmj
How did Neher contribute to our understanding of ionic currents?
Conti and Neher 1980
K+ channel current isolation -> single channel recordings in squid axons
Sighworth and Neher 1980 -> 10^8 -> 10^10 giga ohm seal -> higher resistance = less noise
later this technique allowed for qualification of Na+ channel currents - K+ blocker tetramethylammonium + also use Na+ blocker TTX
> account for voltage and time dependence of Na+ channel conductance
> looking at inactivation
> Hodgkin - Huxley theory / Kinetic theory of Armstrong + Gilly -> final opening step of channel = rate liming in channel activation
How did Aldrich and his colleagues contribute to our understanding of potassium channels? (4 methods)
- first applied trypsin, a more specific proteolytic enzyme, to the cytoplasm face of detached membrane patches contains Shaker channels and found the rapid inactivation was removed
- mutated Shaker to remove 20 amino acids from the amino terminal of the protein
=> resulting channels activated normally but did not inactivate, suggesting that this amino-terminal region constitutes the ball - Shortened and lengthened portion of the proton connecting the ball to segment S1 -> changed the rate at which the channel s inactivate, which is consistent with the idea that this portion constitutes the chain
- Mutated Shaker , lacking the 20 terminal amino acids, was expressed in oocytes => a synthetic peptide corresponding in sequence to these 20 amino acids was able to block the channel when it was added to the cytoplasmic face of detached membrane patches.
What was the conclusion of Aldrich’s experiments?
Strong evidence that rapid inactivation of potassium channels reflects channel block by a tethered blocking particle that is an integral part of the channel protein, They also emphasise the power of carefully designed molecular mutagenesis experiment in function assignment, It’s not the amino terminus that’s responsible for rapid inactivation -> it’s the intracellular loop between the third and fourth homologous domains that is critical for inactivation -> mechanism seems more complex than classical ball-and chain model.
What did experiments using charybdotoxin demonstrate?
MacKinnon 1989 -> Mutagenesis experiments have demonstrated that scorpion toxin, charybdotoxin, a blocker of the external pore of some potassium channels, interacts with amino acids in the region between TM segments S5 and S6 of Shaker, S5-S6 linker must span membrane because some amino acids in this linker are accessible to potassium channel blocker tetraethyl ammonium, applied from the inside, - originally modelled as a extracellular loop
-> now called the P-domain
-> mutations in this region can change the ion selectivity and conduction properties of Shaker channels
when the P domains of two different potassium channels with different conduction properties are swapped, the size of the single channel currents of the resulting chimeric channels is detained by the small P domain rather than by the remaining, the approximately 500 aa of the channel sequence
-> demonstrates critical role of P domain in potassium conductance and selectivity -> direct structural determination
What were MacKinnon and colleagues able to achieve with X-ray crystallography? (general)
able to obtain protein crystals of purified KcsA protein and determine its 3D structure at high-resolution
What challenges did MacKinnon and colleagues face when using X-ray crystallography?
Although structures of many cytosolic proteins have been solved by these techniques, membrane proteins tend to lose their ordered structures when they are removed from the lipid bilayers an have bene very difficult to crystallise.
Main conclusions drawn from X-ray crystallography:
- The four subunits of the tetramer form an inverted teepee -> apex of which points towards the intracellular side of the membrane.
- Extracellular entry to pore at base of teepee is evident from a view looking down on membrane. It has been established by previous biophysical measurement that the selectivity filter is a narrow region toward the extracellular surface of the membrane.
- 2 potassium ions can occupy the selectivity filter simultaneously with a third in a water-filled cavity deeper in the pore, again in accord with predictions from long ago.
- > confirms many of the predictions from mutational analysis about potassium channel conduction and selectivity
Fishman 1973
> closer look at the H-H relaxation time -> noise component measured in voltage clamp -> potassium ion passage process
two-state conductance model -> open-closed based on H-H framework but eluded that other models could fit the data from patch clamp noise -> needed refinement
What was demonstrated by Armstrong, Benzanilla and Rojas in the 1970’s?
- activation on one hand and inactivation on the other hand must involve different parts of the channel protein
2 channel component responsible for inactivation, the inactivation gate, is a protein domain that must be accessible from the cytoplasmic face of the membrane
- activation gate is not accessible to pronase from the cytoplasmic side and hence must involve a different domain of the protein
What were the methods behind Armstrong, Benzanilla and Rojas experiment?
They perfused the inside of the squid giant axon with pronase, a heterogeneous mixture of proteolytic enzymes, and found that this treatment eliminated inactivation but does not affect activation/ deactivation of the axonal sodium current.
