A very nice cryoelectron microscope image came out of the Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States. Richard K Hite was the corresponding author. These authors used a technique called Cryo Electron Microscopy to visualize CIC7 in actual membranes. 
Getting to know the protein explorations
The RCSB database allows the user to look at various sequence features of their crystal structures of proteins based on UniProt entries. This link is for CIC7-OSTM1 in a membrane. Asparagine/N359 is in a hydrophobic region. The arginine side chain in the second box is very hydrophilic with a net positive charge. “Oil and water do not mix.”
N359 is in a loop between two membrane spanning alpha helices. Membrane spanning alpha helices tend to be very hydrophobic on one side with charged residues on the other for the purpose of interacting with other alpha helices. The image on the right is a more 3D rendition of the the packing of alpha helices about one another in the membrane. Perturbation of one can cause movement of the others.
The one thing we cannot forget when looking at the above images is that CIC7 exists in membranes as a dimer, a unit composed of two monomers. If we include OSTM1, a tetramer. That CIC7 as a cystathionine-β-synthase domain will not be explored in this post.
Pathways of Cl– and H+ conduction and osteoporosis causing mutations
Panel 7A shows the proposed H+ (red) and Cl– (green conductance pathways.  Panel 8 is a map of mutation in the CLCN7 gene associated with osteoporosis.  This structure is available online at RCSB.org for closer examination.
This is a zoom in on the dimer interface. A lavender arrow points to Phe360 that is adjacent to Asn359. The chlorides, green spheres, can be seen in both monomers of the dimer in this image.
One more zoom out brings us to the structure. In this case the OSTM1 members of the tetramer are navy and medium blue. CIC7 members of the tetramer are light blue and yellow.
The N359R mutation is predicted to be more or less at the dimer interface between the two CIC7 subunits and below the OSTM1 cap that is there to protect the transporter from the acidic environment of the lumen of the lysosome. The site is somewhat removed from the phosphatidyl inositol binding site.
The PI3P site is for real 
Two ligands co-purified with the chicken ggCLC-7 and CLC-7/OSTM1: ATP and PI3P, a phosphatidylinositol (PI) lipid species enriched in endolysosomal membranes. PI3P constitutes between 0.1% and 0.5% of the total PI content in cells according to references cited by these authors. The authors could not find evidence of PI3P regulating ion transporters, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is potent modulator of ion transport proteins that this post will not get into. They modeled in a PI(3,5)P2 lipid into the binding site in the CLC-7/OSTM1 structure to gain insights into its effect.
The phosphatidyl inositol phospholipid
- The first position of the glycerol backbone is occupied by a saturated fatty acid.
- The second position is occupied by a fatty acid with several double bonds, i.e. an unsaturated fatty acid. These fatty acids are prone to oxidation.
- The inositol group is on the third carbon of the glycerol backbone. The inositol can have a variable number of phosphates attached to the numbered ring. The phospholipid found bound to the CIC7/OSTM complex had only a phosphate attached to the 3rd carbon.
When turning off CIC7, there is something special PI(3,5)P2
Hilton and coworkers of the Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke knocked out the kinase responsible for adding a phosphate group to the 5th position of inositol: PIKfyve kinase. They used a variety of cultured cells to perform these experiments. Depleting PI(3,5)P2 caused lysosomal hyperacidification, primarily via an effect on ClC-7. This interaction was also shown to be important for size regulation.  PI(3,5)P2 inhibition of ClC-7 appears to be specific: neither PI3P nor PI(4,5)P2 yielded detectable inhibition of the ClC-7 currents. As an additional note, PI(3,5)P2 provides a platform for TORC1 signaling from lysosomes.
The next post tries to make sense of the phosphatidyl inositol family of phospholipids, membrane vesicle trafficking, and CIC7 mediated H+ and Cl– transport. Skeletal muscle may be particularly vulnerable due to it’s high reliance autophagy. Click on this link for the continuing story.
- Schrecker M, Korobenko J, Hite RK. Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1. Elife. 2020 Aug 4;9:e59555. PMC free article
- Leray X, Hilton JK, Nwangwu K, Becerril A, Mikusevic V, Fitzgerald G, Amin A, Weston MR, Mindell JA. Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance. Elife. 2022 Jun 7;11:e74136. doi: 10.7554/eLife.74136. PMC free article
- Jin N, Mao K, Jin Y, Tevzadze G, Kauffman EJ, Park S, Bridges D, Loewith R, Saltiel AR, Klionsky DJ, Weisman LS. Roles for PI(3,5)P2 in nutrient sensing through TORC1. Mol Biol Cell. 2014 Apr;25(7):1171-85. doi: 10.1091/mbc.E14-01-0021. Epub 2014 Jan 29. PMC free article
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