Experiments 2 Flashcards
What did Nernst do and when?
1887
Equation that relates thereduction potentialof an electrochemical reaction (half-cellorfull cellreaction) to thestandard electrode potential,temperature, andactivities(often approximated by concentrations) of the chemical species undergoing reduction and oxidation.- different ions have different equilibrium potentials
What did Rakowski do and when?
1989
dialysed and voltage-clamped squid axon
Na,K-ATPase -> measured the ouabain-sensitive current and Na+ fluxes.
What were the conclusions drawn from Rakowski’s experiments?
Their results indicated that the Na/K exchange ratio was precisely 3: 2 and remained constant over a large range of inside and outside N a + and K + concentrations and membrane voltages.
How did Goldshleger’s results confirm Rakowski’s?
confirmed by Goldshleger simultaneously measured Na+ flux and electrogenic activity of Na,KATPase isolated from pig kidney and reconstituted in proteoliposomes. Over large ranges of cytosolic Na+ activity (2-50 mM), pH (6.5-8.5), and ATP (1-1000 JLM) concentrations, they found that the classical 3 Na+:2 K + ratio was maintained.
What did Apell do and when?
1989
Using purified Na,K-ATPase isolated from rabbit kidney medulla and reconstituted in proteoliposomes
Conclusions from Apell’s experiments:
- steep voltage-dependence at high negative membrane potentials, but a voltage independent pump rate between -1 00 and 0 m V. Although apparent saturation of the pump current activity might have many other causes than intrinsic kinetic properties of the pump, the differences of I-V curves obtained in excitable tissue and epithelial cells suggest the possibility that the various Na,K-ATPase isoforms have different voltage sensitivities.
- A steep voltage-dependence in the physiological range of membrane potentials indicates an appreciable control of the Na,K-ATPase activity by the membrane potential.
What did Takeyasu do and when?
1990
Molecular cloning
- > mRNA from tissue is used as a template fro reverse transcriptase, cDNA -> select subunit -> oligonucleotide probes (hybridisation, primers, DNA polymerase)
- > Once functional domains are identified, the fine mapping of the amino acids required for function can be achieved by site-directed mutagenesis
- > site-directed = positional cloning -> cloning at different positions of shaker locus to see how we alter the potassium current -> look for homologous proteins by fundamental oligonucleotide hybridisation
Conclusions from Takeyasu’s experiments:
Sodium potassium exchange pump maintains a high conc. of K+ inside and high Na+ outside -> maintain rmp
Outline experiments by Furshpan & Potter
1968
- crayfish.
-> passed a depolarising current into the nerve and found that the muscle depolarised but then when they hyperpolarised the presynaptic nerve and although the response was small they got a hyperopolarising current in the muscle.
-> when they hyper polarised the muscle, they found they got a massive hyper polarisation in the nerve.
=> current can flow in both directions across the nmj of the crayfish even though it flows better in one direction, the fact that current flowed in both directions meant this was the first electrical synapse to be discovered.
-> found low resistance junctions between cells in embryos and tissue culture
Name 2 studies using dye transfer which studied gap junctions:
Andrew and colleagues 1981 - hippocampus
El-Fouly, Trosko and Chang 1987
Conclusions of dye transfer experiments
- Dye-injection experiments suggest a maximal functional pore size for connection channels ~1.5 nm this means that coupled cells share their small molecules but not macromolecules. This cell coupling has important functional implications.
- Selectivity - use different fluorescent dyes to look at permeability of different gap junctions to different ions
> We can block gap junctions by using lipophilic agents probably a mechanism of per oxidation + basically creating ischemic conditions -> can selectively block gap junctions (but not too selectively) - Conduction
most connexins are phosphoproteins and are thus sensitive to the action of protein kinases and phosphatases
> Phosphorylation of connexin molecules affects the conductance and open time of the channels -> reaction to this depends on subtype
Name 3 studies which used patch clamp to study gap junctions
- > Brink 1989
- > Takens-Kwak & Jongsma 1992 - coupling/ uncoupling
- > Trexler, Vennet & Bargiello - voltage gating and permeation
Conclusions drawn from patch clamp studies on gap junctions:
- Gap junctions are gated by transjunctional voltage:
- open and close in discrete steplike transition between states
- open time is sensitive to voltage
primarily sensitive to voltage differences between coupled cells but much less to cells membrane potential themselves
> reduced when either cell of a pair is depolarised or hyperpolarised / tranjunctional potential increases
even when junctional channels are closed by voltage - the remaining or residual conductance can be as high as 40% of max (Cx43) or as low as 0 (Cx46)
> residual conductance may be due to conformational change in gap junction channels - -> transjunctional voltage shifts most junctional channels from a main state to substate behaviour
What did Ruppersburg and his colleagues do and when?
1990 - they investigated the formation of heteromultimeric potassium channels by using rat brain potassium channel forming proteins (1 + 4). Inserted the homomultimeric channels and heteromultimeric channels into HeLa cells. RCK 1 and 4 (not-sensitive) have different TEA sensitivities and RCK 1+4 is partially sensitive. They were then able to demonstrate by cell-attached patch clamp recordings with TEA applied to the channel that its properties of electrical conductance were different -> it’s a different channel
What did Takahashi and his colleagues do (mgluR) and when?
1996 - Investigated the mechanisms underlying mGluR mediated presynaptic inhibition =>
suppression of a presynaptic calcium conductance, augmentation of potassium conductance and inhibition of exocytotic machinery downstream of calcium influx
1. Identify Calcium channel - isolate calcium current with TEA and TTX
-> Patch clamped post-synaptic side -> synchronous releases from presynaptic side
- current started flowing at -20 mV so they knew it was high voltage
-> use blockers - mostly P-type blocked
2. Figure out which mGluR’s are involved - L-AP4 blocker suppresses Ca2+ current in mGluR4-8
3. Open probability is affected by antagonist -> IV curve shows that what is changed is the ability of channel to open
