Palmitoylation and regulation of divalent cation transport by TRPM7 and TRPM6

Gao, Xing (2022) Palmitoylation and regulation of divalent cation transport by TRPM7 and TRPM6. PhD thesis, University of Glasgow.

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Magnesium regulates numerous cellular functions and enzymatic reactions, and abnormal magnesium homeostasis contributes to vascular dysfunction and the development of hypertension. The transient receptor potential melastatin 7 (TRPM7) is ubiquitously expressed and regulates embryonic development and pathogenesis of several common diseases. It is also a key player in cardiovascular magnesium homeostasis, cardiac fibrosis, and angiotensin II-induced hypertension. The TRPM7 integral membrane ion channel domain regulates transmembrane movement of divalent cations, primarily Ca, Mg and Zn, and its kinase domain controls gene expression via histone phosphorylation. Mechanisms regulating TRPM7 are elusive. TRPM7 not only localizes on the cell surface where it controls divalent cation fluxes but also exists in intracellular vesicles where it controls zinc uptake and release. Palmitoylation is a dynamic reversible posttranslational modification, which regulates ion channel activity, stability, and subcellular localization. We found TRPM7 is palmitoylated at a cluster of cysteines (Cys1143, Cys1144 and Cys1146) at the C terminal end of its TRP domain in multiple cell types. Palmitoylation controls the exit of TRPM7 from the endoplasmic reticulum and the distribution of TRPM7 between cell surface and intracellular pools. Using the Retention Using Selective Hooks (RUSH) system, we arrested TRPM7 in the Golgi and manipulated its palmitoylation with 2-boromopalmitate (2-BP). Pharmacological reduction of TRPM7 palmitoylation reduced its delivery to the cell surface membrane when it was released from the Golgi. we discovered that palmitoylated TRPM7 traffics from the Golgi to the surface membrane whereas non-palmitoylated TRPM7 is sequestered in intracellular vesicles. In addition, we engineered chimeric forms of TRPM7 in which the palmitoylation sites were replaced with the analogous region of TRPM2 or TRPM5, which do not contain cysteines. It also concludes that inhibiting palmitoylation of TRPM7 results in reduced TRPM7 abundance on cell surface. We identified the Golgi-resident enzyme zDHHC17 as responsible for palmitoylating TRPM7 and find that TRPM7 is de-palmitoylated by some acyl-thioesterases post-Golgi and re-palmitoylated by plasma-membrane-resident zDHHC5. The close homologue TRPM6 is also palmitoylated on the C-terminal side of its TRP domain. To investigate the impact of palmitoylation on TRPM7 ion transport activity, we attempted to measure Mg influx at cell surface and Zn influx in intracellular vesicles, but these two assays were unsuccessful. Using fluo4 to measure intracellular Ca we determined that TRPM7 mediated transmembrane calcium uptake is significantly reduced when TRPM7 is not palmitoylated. In addition, we also measured the relationship between phosphorylation/cleavage of TRPM7 and Its palmitoylation. Phosphorylation and palmitoylation are two independent post-translational modifications of TRPM7. Phosphorylated TRPM7 was palmitoylated to the same extent as total TRPM7. Non-palmitoylated TRPM7 chimeras were less phosphorylated, probably as a result of their reduced abundance at the cell surface. Quantitative proteomic analysis of the protein partners of wild type and non-palmitoylated TRPM7 identified vesicular proteins as more enriched with non-palmitoylated TRPM7, and nuclear proteins more enriched with palmitoylated TRPM7, suggesting cleavage and nuclear translocation of the TRPM7 kinase domain may be influenced by palmitoylation. Our findings illustrate palmitoylation controls ion channel activity of TRPM7 and that TRPM7 trafficking is dependent on its palmitoylation. Palmitoylation of TRPM7 might implicated in control of gene transcription by altering nuclear localization of its cleaved kinase domain. In conclusion, we defined palmitoylation as a new mechanism for post translational modification and regulation of TRPM7 other TRPs.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
Supervisor's Name: Fuller, Professor Will and Touyz, Professor Rhian
Date of Award: 2022
Depositing User: Theses Team
Unique ID: glathesis:2022-83245
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 31 Oct 2022 08:39
Last Modified: 31 Oct 2022 08:42
Thesis DOI: 10.5525/gla.thesis.83245
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