Current and voltage clamping Flashcards
What is the concept of voltage-clamps?
Imposed constant, voltage involves an injected current by the device.
At different EMs, there will be ion flow through channels which would change Em
E.G if depolarised, Na+ will enter… Therefore, this flow is matched by an equal injection by the machine to keep Em constant… So what you measure, is the injected current by the device….
= What is on the current graph is what is injected by the device again to stop the ion flow resulting from the new Em.
What direction do ions flow at different Em voltage clamps?
If measured current is positive, then there will be efflux of K+ or influx of Cl-.
If the current is negative, there will be influx of Na+/outflow of Cl-.
What pharmacological agents block VG Na+ and K+ channels?
TEA blocks VG K+ channels.
= Tetraethylammonium
Tetrodotoxin = blocks VG Na+ channel.
= TTX binds to channel, to prevent ion flow.
What are the different patch clamp configurations?
To measure global currents from all open channels:
Use a whole-cell recording
To record unitary currents (Single channel):
Cell-attached.
Inside-out
Outside-out.
Describe the unitary current patch clamp configs?
Outside-out:
Breaking of membrane, which re-anneals, with the extracellular domain of channel accessible (facing bath from solution)
= can change extra.cellular conc of ions + and agents acting on channel exterior…
Inside-out:
Tearing off channel from membrane, so cytoplasmic domain is accessible by bath solution/facing away from pipette.
= Able to modify second messengers in bath solution + conc. of ions…
Cell-attached:
Using mild suction - keep tight contact with pipette and membrane, without destroying PM.
= Can measure/change extracellular ion conc.
=More physiological conditions.
= Single channel possible/not always.
How many states do sodium and potassium channels have?
Closed, open, inactivated for sodium.
Closed to open when Em rises, become inactivated with time, then will return to closed conf. when Em decreases.
Closed and open for potassium.
Become open when Em rises, then close when Em reduces.
How do I/V curves look for Potassium channels?
Until the threshold is met, there is no current flow…. When threshold met, there is increasing outward current as Em increases.
Because the threshold for the channel is more positive than Ek/Erev, When Em is below the threshold, there is no reversal of ion flow direction, since the channel is closed…
What does I/V curve look like for sodium channels?
Above the threshold, there is constant increasing current as Em increases.
Until Erev/Ena when direction of current flow flips from inward to outward.
How to ensure that a single channel is conductive to just one ion?
Measure I/V curve for the suspected ion. Plot graph to find Ek/Erev.
Then measure I/V curve when intracellular and extracellular conc of ion is equal (removing chemical gradient).
= E’ ion / E’ rev of ion will shift to 0mV (As no chemical gradient, and at 0mv there is no electrical gradient) = if it doesn’t shift to 0 then the channel must be conductive to something else too!
Why are Na+ channels faster to open than K+ channels?
There is a delay in activation of K+ channels compared to Na+ channels.
The conformational changes caused by voltage sensing are slower in K+ channels than in Na+ channels - introducing a delay to ion flux.
What is the structure of VG Na+ channels? (Domains and segments)
a complex of 3 glyciproteins:
Alpha subunit is ion channel with two auxilary beta subunits.
The alpha subunit is composed of 4 domains, 1-4.
Each domain is formed from 6 TM segments and an intracellular P loop between S4 and S5.
The segments are folded such that the 4 P loops face each other - to form the central ion channel
What forms the selectivity filter in Na+ channels?
The 4 intracellular P loops (all facing eachother) between S4 and S5, in each of the 4 domains of the Alpha subunit.
= For ion to flow, must be sufficient size and appropriate charge to interact with AAs in P loops .
What are the voltage sensors in Na+ channels?
S4 segment contains voltage sensors - which are positively charged residues.
When the cell is more depolarised, the intracellular conditions are more +ve and the +ve residues in voltage sensor are repulsed - this makes channel more likely to be open.
How do VG Na+ channels inactivate?
Within the alpha subunit, there is a cytoplasmic loop between domains 3 and 4.
IFM AA sequence, physically blocks the channel, particularly F, phenylalanine.
= whilst channel still open.
Requires EM to decrease and conf change to form closed conformation.
= Responsible for absolute refractory period…
What is the structure of VG K+ channels?
4 identical alpha subunits (tetramer of identical alpha subunits)
