Flashcards in CLCs in organelles and acidification disorders Deck (53):
What are the examples of plasma membrane cl channels ?
CLC-1,2 Ka and Kb
-2 is more of an inward rectifier than an outward rectifier
not particularly voltage gated
What are examples of vesicular cl-/H+ exchangers?
these are not channels
Why are lysosomes and endosomes useful to look at when looking at the CLC family?
lysosomes and endosomes are important because you can change the environment inside the organelle compared to the cytosol
Why is vesicle/organelle acidification important ?
- maturation and membrane traffic(e.g. recycling)
- accumulation of neurotransmitter or hormone
- dissociation of ligand from receptor (endocytosis)
important in both exocytic and endocytic vesicles
What is an examples of vesicular endocytosis ?
endocytosis in kidney epithelial cells
- receptor binds substances at surface
- this is then endocytosed and the substance needs to be dissociated from the receptor and then the receptor is recycled
- this process is very important in the kidney proximal tubule, cells to reabsorb substances back into the body
Why is vesicle/organelle acidification important for lysosomes?
important in lysosomes for
- hydrolases (enzymes)
- lysosomes contain many enzymes to digest substances- they have a low pH to help digest substances and also activate enzymes
What does vesicle/organelle acidification require?
- proton-ATPase pump= primary transporter for acidification = active transporter - pumps protons against their electrochemical gradient while using ATP
- ion shunt/leak to dissipate charge= passive meembrane conductance, prevents the build up of proton charge, leaky membrane dissipates charge, either remove cations or make membrane leaky to anions so they can enter
- other channels/transporters
What is Dent's disease ?
hereditary renal tubular disorder caused by mutations in CLCN5
What disorders come under X-linked hypercalciuric nephrolithiasis?
- Dent's disease
- X-linked recessive nephrolithiasis - kidney stones
- X-linked recessive hypophosphatemic rickets
-low molecular weight proteinuria
variations of a single disease with varying severity "the dent's disease complex"
What do most cases of dents disease involve?
loss of function CLC-5 and suggest a defect in endocytosis in the proximal tubule - about 60-70% of mutations
Why does protein occur in the urine?
because it is not being endocytose back into the kidney through epithelial cells from the primary urine
- can lead to complete kidney failure after so many years
in rat kidney where are most of the CLC-5 channels expressed ?
most of the CLC-5 channels are expressed close to the apical membrane of the epithelial cells, the same place as the H+-ATPases
it is also present in the same area as where endocytosis takes place because it is colocalised with villain and beta-2 micro globulin which are low molecular weight proteins that are filtered and then take up by endocytosis by PCT so you dont lose them in your urine
What genetic locus was CLC-5 discovered?
discovered at the genetic locus linked to dent's disease (proteinuria, kidney stones, kidney failure)
What is CLC-5s protein sequence like ?
it is homologous to known CLC chloride channels
Where is CLC-5 localised to ?
localised to subapical vesicles in proximal tubule and colocalises with H+-ATPase
Where have CL- channels been identified?
identified in fused vesicles from renal cortex, important for maintaining electroneutrality
What has CLC-5 proposed function been ?
proposed to provide Cl- shunt conductance
- build up of H+ in vesicle lumen, attracts Cl- to enter to prevent build up of charge
- however it was assumed initially that CLC-5 was a chloride channel
What might be the consequence of CLC-5 loss of function?
impaired endosomal acidification ?
impaired reabsorption of amino acids, glucose and phosphate?
impaired recycling of endocytose membrane proteins : receptors and transporters
What is megalin/cubulin complex?
it is a multiligand receptor - binds to low molecular weight proteins (e.g hormone and vitamin carrier proteins)
= low molecular weight receptor/scaveneger receptor
- fairly non-specific, binds any kind of protein in the primary urine - endosomal acidification will occur to enable receptor to be recycled back to the membrane
What happens when you get loss of CLC-5 expression at the apical membrane ?
brush border and potentially impaired recycling of receptors
using immunofluroersnce what was different between WT mice and CLC5 KO mice?
there was less immunofluoresnce for vitamin D binding protein and retinol binding protein in the KO mice as less endocytosis has occurred and therefore these substances are not being reabsorbed back into the body
- therefore lack of vitamin D means, rickets disease can occur
less immunofluoresnce of megalin and clubbing possibly due to less recycling due to decreased endosomal acidification - then they are shunted down lysosomal pathway to be degraded instead od recycled
What are the consequences of impaired hormone and ion reabsorption in the proximal tubule ?
defective calcium and phosphate homeostasis
vitamin D loss that can lead to rickets
accumulation of calcium and phosphate in tubules can lead to kidney stones (formed from calcium oxalate and phosphate)
parathyroid hormone in tubules thought to stimulate internalisation of NaPi-2a transporter
What happens in the PT cells in normal mice ?
apical membrane of pT is where endocytosis occurs
- substances are absorbed and form late endosomes that fuse with lysosomes
What happens in the PT cells in dents disease /?
there is insufficient recycling so there are fewer receptors at the surface membrane therefore less endocytosis so more substances, ions, low molecular weight proteins in urine
What are the different mutations in CLC5 dents mutations >?
many of the mutations occur between the interface of the 2 subunits
CLASS 1: cause retention of the protein in the ER which ends up being degraded in the ER
CLASS 2: end up in the correct place but they are non-functional, they have no transporter activity at all
CLASS 3: mis-targeting of protein- not in the correct organelle at the correct time
What is the inefficient theory for how CLC-5 works ?
it is colocalised with H+-ATPase
- it is thought that 3 ATP molecules are used to move 3 H+ into the organelle by the H+-ATPase but then the CLC-5 channel pumps 2 cl- ions in and this means one of the H+ is pumped back out and therefore this is not a particularly efficient prices because 1/3 of the protons leave the endosome in acidification
- this doesn't seem logical when other chloride channels could provide the chloride leake condutance
What does overexpression of clc-5 in HEK cells do ?
it means they can be expressed in the plasma membrane and therefore you can carry out electrophysiological recordings
- outwardly rectifying proteins
What is reconciliation with the CLC-5 shunt hypothesis ?
CLC-5 behaves differently in endosomal membranes compared to plasma membrane - membrane content, protein interaction and ion gradient
tiny, non zero current enough to conduct cl- into endosomes
What are the 2 critical analyses of CLC-5 shunt model ?
1) it is inefficient- losing 1/3 of the energy
2) electrophysiological properties are inconsistent with this role
What is meant by electrophysiological properties are inconsistent with this role of CLC-5 shunt model ?
outward currents (positive charge out and/or negative current into the cell
- when oocytes express clc5 pH dropped near the membrane
- if there was a positive charge build up inside the endosome this would be equivalent to hyper polarisation and clc5 would be inactive
also when clc5 is internalised into the vesicle the cl- side is always facing the cytoplasmic side
Can CLC-5 conduct protons into endosomes in parallel with H+-ATPase ?
experimentally it is very different to distinguish between the 2 especially if cl- influx into endosomes can occur through an alternative pathway
How is endosomal pH regulated?
through the activity of both v-ATPase and CLC-5
How did they measure acidification when CLC-5 was overezpressed in an endosome and what was the outcome ?
attached pHluorin fusion protein intracellularly to vAMP 2
- when clc5 is overexpressed there is increased acidification and greater fluorescence of pHluorin
What happens to the endosome when bafilomycin A1 is present ?
it inhibits H+ATPase and when it is present the endosome has a more alkaline pH
- whereas in the CLC-5 expressing endosome the endosomal pH is more acidic
What happens with these 2 mutants (E211A and E268A)?
transport of the channel is disrupted and it makes it into chloride channel bu removing H+ transport
- they are constitutively active
-neither can enchance acidification indicating that theses channels induce acidification independent of H+ pump
What are the ion dynamics in endosome-lysosome maturation ?
early endosomes may initially have internal negative charge (excess of surface charges) and be rich in NaCl
therefore could cl- and na+ extrusion drive h+ into endosomes independent of H+-ATPase
What effect on mice does knocking in E211A CLC5?
Chnages CLC5 into a constitutively active chloride channel
- disrupts acidification in vesicles
- causes deficient endocytosis
- have normal ATP-dependent endosomal acidification but defective kidney function similar to CLC5-/y ko mice and human dents disease
What is the conclusion?
clc5 mediated 2cl-/H+ exchange rather than cl- conductance is crucial for kidney function
clc5 is not a shunt conductance
H+ efflux from acidic endosomes through CLC5 causes accumulation of Cl- and this is important
What mutations are found in patients with osteopetrosis?
What are the symptoms of osteopetrosis?
What is happening in osteopetrosis?
its defective bone resorption, bones become very dense and this compresses nerves leading to degeneration
- poor bone remodelling
- enlarged, dense bone
- compression of optic nerve lead to degeneration or retinal degeneration
What was seen in clc7 ko mice?
osteoclasts fail to dissolve bone
- osteoclasra are the cells that adhere to bone and release acid and enzymes to dissolve bones
- this is important for remodelling
What is clc7 thought to be important for in osteoclasts?
thought the cl/H transporter is important for efficient H+ pumping at the ruffled membrane of osteoclasts, formed by exocytotic insertion of late endosomes and lysosomes
What are grey lethal mice and what do they do ?
osteopetrosis and related symptoms are observed in the grey lethal mouse
the are mice that spontaneously had OST symptoms
carry a deletion of the OSTm1 promoter and exon 1
What do OSTM1 defects in huamns cause?
cause severe recessive osteopetrosis
OSTM1 required for clc7 transport activity and targeting of protein to lysosomes
- ostm1 is an accessory protein for clc7
these proteins have to coassemble for proper functioning of clc7 - function and expression
What is apparent about clc-7 KO mice?
animals are smaller due to poor remodelling
- defects in osteoclasts cant dissolve bones for remodelling causing reduced growth
What were the differences of osteoclasts from WT and clc7 ko when cultured on ivory?
osteoclasts were harvested and cultured and then placed on ivory
-WT: there were pits in the ivory where the osteoclasts would have been residing as this occurs due to them releasing acid and enzymes for secretion
-KO: defect in osteoclasts therefore no pits due to defect in acidification and acid dependent enzymes
In WT what does CLC7 colocalise with ?
colocalises with OSTM1 in LAMP2 containing lysosomes
What happens to CLC7 in R525W mutant ?
OSTM1 and mutant CLC7 are retained in the ER
What happens to CLC7 in L490F mutant ?
OSTM1 and mutant CLC7 are targetted correctly to lysosomes but clc7 is non-functional
What happens to CLC7 in S744F mutant ?
OSTM1 and mutant clc7 are targeted to lysosomes but clc7 functions similar to wt
What is it classified as when CLC7 has an accelerated function ?
classified as a gain of function