lysosomes

TREK1

BL

The featured image comes from ProteinAtlas that has something interesting to say about the organs in which TREK1 is expressed. It is not such a brain protein. According to UniProt.og ” TREK1 is an ion channel that contributes to passive transmembrane potassium transport. Reversibly converts between a voltage-insensitive potassium leak channel and a voltage-dependent outward rectifying potassium channel in a phosphorylation-dependent manner. In astrocytes, forms mostly heterodimeric potassium channels with KCNK1, with only a minor proportion of functional channels containing homodimeric KCNK2. In astrocytes, the heterodimer formed by KCNK1 and KCNK2 is required for rapid glutamate release in response to activation of G-protein coupled receptors, such as F2R and CNR1 (By similarity).”

Sortillin

According o UniProt.org, the SORT1 gene codes for a protein called sortilin functions as a sorting protein in the Golgi to cell surface transport. It is also required for transport from the Golgi to lysosomes …Much of the UniProt entry reads as if it were AI written. UniProt also has an iterative diagram for sortillin that is worth 10,000 AI written words

ote: Localized to membranes of the endoplasmic reticulum, endosomes, Golgi stack, lysosomes and nucleus. A small fraction of the protein is also localized to the plasma membrane. May also be found in SLC2A4/GLUT4 storage vesicles (GSVs) in adipocytes. Localization to the plasma membrane in adipocytes may be enhanced by insulin.

These endosome vesicles take cargo from one location of the cell to another. There are naturally some incredibly complicated nuances of this process that we do not need to understand the truly bizarre story that this membrane bound vesicle sorting molecule can regulate a potassium channel in neurons.

TREK1 is one of many voltage gated K+ channels in the nodes of Ranvier that are responsible for the hyperpolarization part of the action potential. Blocking this hyperpolarization blocks the entry into the resting phase.

Removing and inserting TREK1 channels: Sortilin 1

This image was derived from a summary figure from Mazella 2010, a screen capture from the UniProt.org entry on Sort1, and published dequences for spadin and PE 22-28

Right: The signal sequence directs a new protein from the ribosome, where it is made, to the endoplsamic reticulum where it is modified. The propeptide is what keeps the protein inactive until an activator enzyme comes in. Middle The SORT1 seqeunces corresponding to the signal and propeptide and been pasted in. Sequences of spadin and PE-22-28 have been pasted in to prove that pro-peptides are being used as a drug. Right: Spadin, the propeptide or sortillin, is used to send TREK-1 to early endosomes.

This post is going to refer to both the Mazella and the Djillani publications as work from these authors until switching to the connection with the CLCN family of Cl–/H+ antiporters.

Spadin

Mazella J, Pétrault O, Lucas G, Deval E, Béraud-Dufour S, Gandin C, El-Yacoubi M, Widmann C, Guyon A, Chevet E, Taouji S, Conductier G, Corinus A, Coppola T, Gobbi G, Nahon JL, Heurteaux C, Borsotto M. Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design. PLoS Biol. 2010 Apr 13;8(4):e1000355. PMC free paper

PE-22-28

Djillani A, Pietri M, Moreno S, Heurteaux C, Mazella J, Borsotto M. Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity. Front Pharmacol. 2017 Sep 12;8:643. PMC free paper

PE-22-28 is a peptide derived from spadin, a protein secreted by sortillin.

This image came from a Peptide Science post on PE 22-28. Note the multiple levels of TREK1 activation in this cartoon that are mentioned in other sites.

For the most part the Peptide Science post discussed how PE 22-28 might be a better treatment for depression than traditional SSRI.

Note that when TREK1 is open, the neuron hyperpolarizes tending to shut things down. Turning TREK off would tend to to turn the neuron on.

The authors found evidence of radiolabed spadin the the brains of the animals after intravenous and intraperitoneal injections. PE 22-28 proved superior to other peptide analogs in numerous tests involving depression like behavior. While very encouraging for patients with depression, this study did not really address other potential sites of TREK1 expression outlined in the feature image of this post except the andrenal gland.

These authors had demonstrated that the deletion of the TREK-1 gene reduced the
hypothalamic-pituitary-adrenal (HPA) axis. IP spadin reduced corticosterone levels 30 min after a 10 min tube restraint, a paradigm known to activate the HPA axis.

Retrograde transport, sortilin, and CLCNs

All three of the publications came from Stephane Lefrancois’s laboratory at McGill University.

Players

  • Rab7a is a protein involved in the endocytosis of membrane bound proteins and their trafficking of the endosomes to lysosomes.
  • PLEKHM1, or Pleckstrin homology domain containing family member one, is a binding partner of Rab7a.
  • Retromer is a complex of proteins responsible for recycling of membrane proteins back to the trans Golgi network. Retromer may also interact with sortillin and the mannose-6-phosphate receptor.
  • CLCN5 is the gene that codes for the H+/Cl− exchanger ClC-5. It is expressed in the brain, muscle, intestines, and mediates receptor endocytosis in proximal tubule cells of the kidney. Voltage dependence was mentioned by Wikipedia authors, which makes it particularly interesting when it comes to TREK1 voltage gated K+ matters. Some mutations in this gene cause Dent’s Disease.
  • CLCN3 is the gene that codes for the H+/Cl− exchanger ClC-3. It is expressed in the brain and smooth muscle. Mutations are associated with hypotonia.
  • Mannose 6 phosphate receptor binds lysosomal enzymes bearing phosphomannosyl residues in the Golgi such they are trafficked to the lysosome. The acidic conditions of the lysosome trigger the release such that the mannose-6-phosphate receptor may be recycled back to the Golgi.
  • CLN3 mediates microtubule-dependent, anterograde transport connecting the Golgi network, endosomes, autophagosomes, lysosomes and plasma membrane, and participates in several cellular processes such as regulation of lysosomal pH, lysosome protein degradation, receptor-mediated endocytosis, autophagy, transport of proteins and lipids from the TGN, apoptosis and synaptic transmission

CIC5 binds to sortiln [1]

CIC5 and sortilin were expressed in in the HeLa cell culture system. [1] Antibodies to one were found to immunoprecipitate the other. Binding was observed at acidic ( pH 5) and pH 7 neutral pH. CIC5 was concluded to not be a cargo protein because cargo of sortilin is released under acidic conditions. Through a series of genetic manipulations in HeLa cells, CIC5, but not CIC1, was found to be necessary to recruit retromer to endosome membranes. CIC5 was found to recruit and activate Rab7.

Figure 10 “When the cargo-loaded lysosomal sorting receptor (green line) arrives at the endosome (Step 1), the change in pH causes a dissociation of the cargo from the receptor that subsequently leads to interaction with CLN5 (Step 2). This enables the recruitment and activation of Rab7 and the recruitment of retromer (Step 3) for recycling to the Golgi compartment, where it can interact with more cargo”

What exactly does this model mean for a K+ channel such as TREK that is modulated by N-terminal peptides of sortilin also known as spaden and PE22-28?

CIC3 binds to sortilin and Rab7 [2]

The second publication was similar to the first except instead of using immuno precipitation the authors used bio luminescence resonance energy transfer (BRET)between the proteins of interest fused with fluorescent proteins. An added twist was the use of disease causing mutations in the CLCN3 gene.

  1. CIC3 interacts with sortilin and Rab7A but
  2. CIC3 is not required for recruitment of Rab7A to membranes but.
  3. CIC3 is required for Rab7A interaction with the retromer
  4. CIC3 interacts with the retromer and sortilin
  5. CIC3 regulates the stability of sortilin and CI-MPR. A compound called cycloheximide was used to shut off new protein production so as to be able to determine degradation rates in CLCN3 and Rab7A knock out cells.
  6. CIC3 is required for the efficient degradation of the epidermal growth factor receptor when it is endocytosed.
  7. CIC3 modulates EGFR degradation by modulating sortilin and Rab7A-PLEKHM1 interaction.
  8. CLN3 is a endolysosomal integral membrane protein whose mutations also
    result in Neuronal Ceroid Lipofuscinosis

Tying CLN3 and CIC5 together [3]

Note: CLN3 is not the same proteins as CIC3, the product of the CLCN2 gene!

  1. In CLCN5 knock out HeLa cells, Rab7a palmitoylation is reduced and
  2. Rab7a interaction with sortilin/retromer in these same CIC5 lacking cells
  3. More sotrtilin is degraded in these same CIC5 lacking cells.
  4. CIC5 modulates CLN3 interactions.
  5. Removal of CIC5 is required for efficient degrdation of the epidermal growth factor and receptor complex.
  6. CIC5 is needed for regrograde transport of lysosomes to the
  7. Lack of CIC5 inhibited phagosome/lysosome fusion.

CLCN and sortilin references

  1. Mamo A, Jules F, Dumaresq-Doiron K, Costantino S, Lefrancois S. The role of ceroid lipofuscinosis neuronal protein 5 (CLN5) in endosomal sorting. Mol Cell Biol. 2012 May;32(10):1855-66. PMC free article
  2. Yasa S, Modica G, Sauvageau E, Kaleem A, Hermey G, Lefrancois S. CLN3 regulates endosomal function by modulating Rab7A-effector interactions. J Cell Sci. 2020 Mar 16;133(6):jcs234047. PubMed free article
  3. Yasa S, Sauvageau E, Modica G, Lefrancois S. CLN5 and CLN3 function as a complex to regulate endolysosome function. Biochem J. 2021 Jun 25;478(12):2339-2357. PubMed free article

TREK1 and CIC5

What could possibly be the implication for TREK1 in lysosomal storage disorders involving CIC3, 5, 7? Do mutations in these antiporters reduce the amount of sortillin recycled in the pathways outlined in the Lefrancois publications? If so, does this affect TREK1 modulation? Would the PE 22-28 peptide help matters?

Diagram of the pathways that intersect endosomes in the endocytic pathway of animal cells. Examples of molecules that follow some of the pathways are shown, including receptors for EGF, transferrin, and lysosomal hydrolases. Recycling endosomes, and compartments and pathways found in more specialized cells, are not shown. from Wikipedia and three endosomes from the summary figure of ref {1]

The modified Step 3 endosome of the Lefrancois Fig 10 [1] pathway has not been added. Is there a cargo release of TREK1? Which way does the pathway go? Retrograde to the Golgi or to the lysosome?

Going back to the featured image. TREK1 is not only a brain K+ channel. Is this channel that binds to sortillin get affected more than ordinary growth hormone receptors like EGFR in lysosomal storage disorders involving CIC3 and CIC5?

Published by BL

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