Introduction Flashcards
What are channels?
GATED pores in the membrane
What happens when channels open?
What does this generate?
Ions flow through them
Get CONDUCTANCE
Generates a CURRENT
What happens when the channels are closed?
No ions through
No conductance
No current
What is important about the opening of the channels?
Timing of the opening
Disruption can have an adverse effect on the normal physiology of nerves and muscles
How did ion channels use to be classified?
Through 3 main features:
1) Selectivity
- What is the main ion that moves through the pore?
2) Gating
- What is needed to open the channel
3) Regulation
What could be in control of gating the ion channel?
Voltage
Ligand
Mechanical
What could regulate the channel?
ATP
G proteins
Ca2+
Now, how are ion channels classified?
By the there MOLECULAR STRUCTURE (amino acid sequence and structure):
- Put into groups where there are high similarities in the amino acid sequence
Describe the distribution of K+ channels
What does this mean for mutations?
What other channels is this true for?
Many K+ channels with high similarities in amino acid sequence
But they are DIFFERENTIALLLY EXPRESSED in different tissues
If have a mutation effecting one K+ channel - will impact ONLY the cells where that channel is expressed
True for any other type of channel
When a channel is open, what does it drive the membrane potential to?
The Nerst (reversal) potential for that channel
What is the nerst potential?
- Potential where there is no net current flow across the membrane for that ion
- Ions moving in balance ions moving out
What is Rt/Zf at BODY TEMPERATURE?
61.5
What is Rt/Zf at ROOM TEMPERATURE?
58.2
What is the intraceullar [K+]?
150mM
What is the extracellular [K+]?
5mM
What is the Ek?
What does this show about the membrane permeability at rest?
-89mV
At rest - membrane is -70mV, showing high permeability to K+
BUT, must be other channels open in the membrane (drive the membrane potential to their nerst potential) as the potential is not exactly -89mV
Why does the Nerst potential of the K+ channel sit slightly more positive than the Nerst potential for the K+ ion?
K+ channels have an Na+ leak
Reversal potential of a channel is dependant on ALL the ions that move through the ion channel
What is the extracellular [Na]?
150 mM
What is the intracellular [Na}?
15mM
What is the Ena?
+66mV
Why is the nerst potential of the Na+ channel slightly hyper polarised compared to the nerst potential for the Na+ ion?
Na+ channel has a K+ leak
Nerst/reversal potential of a channel is dependant on all the ions that move through the channel
What is the resting potential of the membrane?
What does this show?
-70mV
Shows that the membrane is more permeable to K+ than is to Na+, as the membrane potential sits closer to Ek
(50x more permeable to K+)
How much more permeable to Na is the membrane at the peak of the AP?
5 x more permeable than K+
What is the intracellular [Cl-] concentration?
6mM
What is the extracellular [Cl-] concentration?
100mM
What is the Ecl?
-87mV (very close to Ek = - 89mV)
Why is looking at the nerst potential for a cell insufficient to be SURE of the ion channel that is open in the membrane?
What can be used to back up the ideas made using the Nerst eqn?
Nerst potentials of different channels can be very close together - unsure which one is open
(Ek = -89mV, Ecl = -87mv)
Patch clamp technique used to back up
What is the current equation?
I = N.Po.g. (Vm-Ei)
N = number of channels Po = open probability of the channels g = a constant (open channel conductance) Vm-Ei = driving force of an ion
What is the basis of the patch clamp technique?
Change and clamp the Vm (potential)
Measure the current (see how change in Vm effects current)
How is the current through a channel regulated?
How can these be changed?
- Number of channels (change by membrane shuttling)
- Open probability of channels (change by ATP,GTP,phos, Ca2+ etc.
- Potential (change through opening/closing different channels)
What does changing the membrane potential change?
Changes the DRIVING FORCE
What is the current recorded with the patch clamp technique called?
What is this?
Itotal (total current across the WHOLE membrane)
What is Itotal equal to?
What is this in a cell with Na channels and K channels only?
The SUM of the currents through the open channels
Itotal = Ina + Ik
What is each of the current in the eqn Itotal = Ina + Ik described by?
Ina or Ik = N.Po.g. (Vm-Ei)
How can the different channel types be found in the membrane using the patch clamp technique?
1) Itotal (current through all the channels in the cell) measured when clamp the potential of the cell
2) Itotal = I1 + I2 +I3 etc….
3) Each I is described by I = N.Po.g. (Vm-Ei)
What does each horizontal line on the whole cell current recording represent?
The current recorded at each different potential (change in Vm)
How can the ion channels that are causing the Itotal be pharmologically identified from the whole cell current recordings?
Can add a BLOCKER for a specific channel and see if the current decreases
What is an example blocker of K+?
What happens when this is used?
Barium
When used - sends the current flow towards 0 (blocking the K+ channels)
Why are Nav channels difficult to look at?
They are CLOSED at -ve potentials (current= 0)
Activate QUICKLY with depolarisation - giving an increase in current
CLOSE with depolarisation slowly
What are the 3 separate states of configuration of the Na+ channels
1) Closed
- Pore not open
2) Open
- Activated by +ve shift in the membrane potential
3) Inactivated
What triggers the inactivated state of the Na+ channel?
Depolarisation of the membrane
However, this takes longer to occur than activation - happens afterwards
How is the structure of an inactivated Na+ channel different to the closed channel?
In an inactivated channel - pore is open but there is a BLOCKAGE in the channel
Blockage - ball of amino acids
What is shown on the whole cell patch clamp recording for Na+?
Why?
Which channels show the same profile as this?
Increase and then decrease in the recording
Na+ channels first open and then close - due to the ball of amino acids blocking the pore of the channel in the inactivation state
Ca2+ voltage gated channels show the same profile
How can Nav be distinguished from Cav in the whole cell patch clamp technique?
Nav is blocked by TETRODOTOXIN
Cav are not
What is FHEIG?
- Bi-temporal narrowing
- Excess body hair
- Thin upper lip
- Overgrowth of tissues inside the mouth
Caused by:
- GENETICALLY inherited mutation in KCNK-4 (K+ chanenl)
- Single amino acid change in this channel
- GOF mutation
Where is the KCNK-4 K+ channel normally expressed?
In the CNS and PNS
What does over expression of the KCNK-4 K+ mutants cause?
What does this show?
LARGER CURRENTS
Shows that this is a GOF mutation
What technique proved that FHEIG was due to a GOF mutation?
Patch clamp technique
What was it hypothesised about the cause of FHEIG?
- KCNK-4 K+ channel found in the CNS and the PNS (but not ALL the cells)
- GOF –> Excess loss of K+ from the cells within the mutant channel into the interstitial space (where there is normally LOW K+)
- As K+ accumulates here - Ek becomes less negative (depolarised)
- Resting potential more positive
- Neighbouring cells more depolarised - closer to threshold potential
- More likely to fire action potentials (normally wouldn’t)
Why is K+ accumulates in the interstitial fluid does the Ek become less negative?
Ei = 61.2 x log ( [K]out/ [K]in)
Higher [K]out = higher ( [K]out/ [K]in) ratio –> when ‘logged’ = more positive
