Cl- channels and transporters formed by the CLC protein architecture

Question 1

After discovery CLC-1 what was thought?

Answer 1

thought that the members discovered after CLC-1 would be analogous

Question 2

What were the electrophysiology recordings from CLC-0 channels like ?

Answer 2

low activity at negative potentials but it increases as the potential becomes more positive/depolarised

Question 3

After mutating the “gating glutamate” what did the recordings show ?

Answer 3

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

Question 4

What does neutralisation of the gating glutamate do ?

Answer 4

it removes the voltage dependent channel closure of CLCs

Question 5

What did electrophysiology recordings of WT CLC-0 channels in pH 5 show ?

Answer 5

increased proton concentration
- increased protonation of the gating glutamate residue which causes the same effect as it being mutated - neutralises glutamate

Question 6

What did single channel recordings of a mutant CLC-0 channel show?

Answer 6

hyperactive, rarely goes into closed state because the glutamate is neutralised so it is not gating the external binding site

Question 7

What is the CLC pore domain like ?

Answer 7

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

Question 8

What is important about the sequence in the conducting pathway ?

Answer 8

amino acid sequence G——P conserved in humans clc-1, clc-0 and E.coli

Question 9

What are the 3 binding sites for anions ?

Answer 9

external
central
internal

Question 10

What is the WT E.coli CLC channel like ?

Answer 10

• 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

Question 11

What is M E.Coli CLC channel like (E148Q)?

Answer 11

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

Question 12

Why is CLC channel activity increased at low pH ?

Answer 12

due to increased protonation

Question 13

Using E.Coli how did they prove that CLC-1 was not purely conducting Cl- ?

Answer 13

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

Question 14

What happened in E.Coli CLC-1 channels when the intracellular and extracellular concentrations of cl- are analogous ?

Answer 14

when the pH changes on either side of the membrane the current-voltage relationship changes indicating that the channel is conducting H+

Question 15

After further experiments what was shown ?

Answer 15

demonstrated the property consistent with 2Cl-/1H+ counter-transporter

Question 16

What is CLC-ec1?

Answer 16

it is a secondary active transporter

Question 17

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

Answer 17

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

Question 18

How did they prevent the charge build up in liposomes occurring and preventing transport?

Answer 18

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

Question 19

What is FCCP ?

Answer 19

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

Question 20

What are the conclusion from the experiments on CLC-ec1?

Answer 20

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

Question 21

Where are CLC-4/5 members normally present ?

Answer 21

mammalian members are not normally present in the plasma membrane they are present in intracellular organelles, endosomes
- organelles that are normally acidic

Question 22

How do you obtain expression of CLC-4/5 in the plasma memebrane ?

Answer 22

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

Question 23

What was seen from electrophysiology experiments of CLC-5 in the plasma membrane ?

Answer 23

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

Question 24

Which channel demonstrate no proton transport?

Answer 24

CLC-0,2 or Ka

Question 25

What does mutation of the gating glutamate do ?

Answer 25

it abolishes proton transport and voltage dependence

- makes it a chloride ion channel, constitutively active

Question 26

What does activation of CLC4/5 do ?

Answer 26

causes acidification of the extracellular space

Question 27

Why are CLC-0/1/2 broken transporters?

Answer 27

have a cyclical activity, between inactive states

proton electrochemical gradient is driving the non-equilibrium behaviour - similar to CFTRs

Question 28

What is the proton glutamate ?

Answer 28

it is different from the gating glutamate = faces inwards and is present in all CL-/H+ exchangers but not in the ion channel

Question 29

What happens when the proton glutamate is mutated?

Answer 29

it doesnt change it into a channel
- it is an intracellular proton acceptor
whereas the gating glutamate is on the outside

Question 30

What other state was identified in the structures of another CLC channel and where was the channel from ?

Answer 30

used x-ray crystallographic studies
CmCLC from thermophilic red alga cyanoidioschyzon merolae has a CBS domain
the gating glutamate occurs the CENTRAL binding site

Question 31

What occupies the binding sites in EcCLC WT channels?

Answer 31

in closed state, non-conducting state there are 2 anions occupying the central and internal bindings sites and the gating glutamate is within the external site

Question 32

What sites can the gating glutamate occupy?

Answer 32

it has been shown that it can occupy the external site and also the central binding site

Question 33

What is stage 1 of the kinetic scheme for how the 2CL-/H+e exchanger might work ?

Answer 33

protein with 2 anions bound in the central and internal binding sites and the gating glutamate is in the external binding site, causing the channel to be closed

Question 34

What is stage 2 of the kinetic scheme for how the 2CL-/H+e exchanger might work ?

Answer 34

gating glutamate moves from external site to central binding site and pushes one anion out at the bottom o the channel and this enables one to come into the top external binding site

Question 35

What is stage 3 of the kinetic scheme for how the 2CL-/H+e exchanger might work ?

Answer 35

then the intracellular proton moved into the internal site to neutralise the gating glutamate at the central binding site making it protonated !
this makes it not electrically favourable to occupy the binding site
this therefore causes the gating glutamate to swing out to the extracellular space which allows more anions to enter the ion conducting pore

Question 36

What is stage 4 of the kinetic scheme for how the 2CL-/H+e exchanger might work ?

Answer 36

then the gating glutamate becomes negatively charged again and reenter the external site pushing an anion down

Question 37

With reference to the kinetic scheme of the exchangers what could CLCs do ?

Answer 37

they may not do stages 1 and 2
or
they may do too much of stages 3 and 4
Stages 3 and 4 look like they have an open ion channel pore

Question 38

What are the arguments for extrinsic voltage sensing in CLCs?

Answer 38

• CLC function is dependent on ions binding in the pore and on ionic gradient
• ion binding and movement in the pore is driven by voltage
• therefore proton or chloride ions may be voltage sensor

Question 39

What are the arguments for intrinsic voltage sensing in CLCs?

Answer 39

• some mutations in CLC1 or CLC0 modify voltage dependent activation
• voltage dependent conformational changes detected in a permeation deficient CLC5, analogous to S4 movements giving rise to gating currents