Cl- channels and transporters formed by the CLC protein architecture Flashcards
After discovery CLC-1 what was thought?
thought that the members discovered after CLC-1 would be analogous
What were the electrophysiology recordings from CLC-0 channels like ?
low activity at negative potentials but it increases as the potential becomes more positive/depolarised
After mutating the “gating glutamate” what did the recordings show ?
loss of this conserved glutamate residue caused….
- loss of voltage dependence causing the channel to become constitutively active , dont see rectification at negative potentials
- this implies the glutamate side chain is part of the gating mechanism
gating glutamate is negative at normal pH and it is neutrallised by mutation of another amino acid so it doesn’t act as a gate
What does neutralisation of the gating glutamate do ?
it removes the voltage dependent channel closure of CLCs
What did electrophysiology recordings of WT CLC-0 channels in pH 5 show ?
increased proton concentration
- increased protonation of the gating glutamate residue which causes the same effect as it being mutated - neutralises glutamate
What did single channel recordings of a mutant CLC-0 channel show?
hyperactive, rarely goes into closed state because the glutamate is neutralised so it is not gating the external binding site
What is the CLC pore domain like ?
CLC channels are made up of 2 separate monomer peptides
- each monomer has pseudo symmetry - contain 2 homologous domains similar to CFTR transporters having 2x 6TMD
- 3 pore regions scattered over course of proteins- alpha helices span across membrane but some only just dip in an out
What is important about the sequence in the conducting pathway ?
amino acid sequence G——P conserved in humans clc-1, clc-0 and E.coli
What are the 3 binding sites for anions ?
external
central
internal
What is the WT E.coli CLC channel like ?
- in closed state the glutamate side chain acts as a gate, it occupies the external binding site because it is negatively charged
- there are only anions in the central and internal binding sites
- no conduction occurs as glutamate is present in the external site but once it moves out conduction occurs
What is M E.Coli CLC channel like (E148Q)?
changed glutamate to glutamine
glutamine isn’t negatively charged so it won’t occupy the external anion binding site
there are anions present in all the anion binding sites making it constitutively active
Why is CLC channel activity increased at low pH ?
due to increased protonation
Using E.Coli how did they prove that CLC-1 was not purely conducting Cl- ?
300mM intracellular cl concentration and a 45mM cl concentration the channel has a linear voltage current relationship
- Erev= 30mV and Ecl= 45mV
- this indicates the channel isn’t purely conducting cl- ions because if it did then the Erev would be exactly the same as the chloride equilibrium potential and its not
What happened in E.Coli CLC-1 channels when the intracellular and extracellular concentrations of cl- are analogous ?
when the pH changes on either side of the membrane the current-voltage relationship changes indicating that the channel is conducting H+
After further experiments what was shown ?
demonstrated the property consistent with 2Cl-/1H+ counter-transporter
What is CLC-ec1?
it is a secondary active transporter
What was shown when CLC-ec1 channels were reconstituted into liposomes and put into small container containing small electrodes?
- high intracellular cl concentration and low outside and small proton gradient
idea was that if you could get chloride ions to leave the liposome then the protons should enter the cell and this should cause alkalisation of extracellular fluid
- it was a highly electrogenic process= as protons increase in liposomes it causes an increase charge which will cause transport to stop
How did they prevent the charge build up in liposomes occurring and preventing transport?
used valinomycin which is a potassium ionophore- it was required to dissipate the build up of positive charge in liposomes
- causes the membrane to become leaky to potassium enabling it to leave t prevent too much intracellular positive charge= shunt conductance
enabling chanel activity to continue
What is FCCP ?
a proton ionophore
- allows protons to leak out again down electrochemical gradient
- it stops channel activity
- it is a metabolic poison, causing collapse of mitochonidral proton gradient
What are the conclusion from the experiments on CLC-ec1?
it is a 2cl-/H+ exchange transporter, not a chloride channel
it is not voltage gated
doesn’t have intracellular CBS domains
Cl- gradient can drive protons
Where are CLC-4/5 members normally present ?
mammalian members are not normally present in the plasma membrane they are present in intracellular organelles, endosomes
- organelles that are normally acidic
How do you obtain expression of CLC-4/5 in the plasma memebrane ?
overexpress the gene in a recombinant system such as xenopus oocytes to obtain expression within the plasma membrane
- this allows electrophysiology experiments to be carried out
What was seen from electrophysiology experiments of CLC-5 in the plasma membrane ?
as depolarising pulses were applied outward currents were recorded and acidification occurred
- the pH only started to restore to normal pH once the depolarising stimulus was stopped
Which channel demonstrate no proton transport?
CLC-0,2 or Ka
