Cellular functions of the kinase-coupled TRPM6/TRPM7 channels

Ferioli, Silvia (2018): Cellular functions of the kinase-coupled TRPM6/TRPM7 channels. Dissertation, LMU München: Fakultät für Biologie

Abstract

TRPM6 and TRPM7 are bifunctional proteins containing an ion channel segment covalently linked to a kinase domain. Both proteins function as divalent cation-selective channels highly permeable to Mg2+ and Ca2+, whose activity is regulated by intracellular levels of Mg2+ and Mg•ATP. TRPM7 has been found in all mammalian cells investigated so far, while expression of TRPM6 is restricted to epithelial cells of the kidney, intestine and placenta. TRPM6 and TRPM7 have been proposed to be required for cellular Mg2+ homeostasis. However, the exact role of TRPM7 in the regulation of Mg2+ metabolism remains poorly understood and discussed controversially. Loss-of-function mutations in the human TRPM6 gene give rise to an autosomal recessive disorder called hypomagnesemia with secondary hypocalcemia (HSH). It has been suggested that in epithelial cells TRPM6 functions primarily as a constituent of heteromeric TRPM6/M7 channel complexes, but this concept has not been thoroughly examined. Therefore, the main goal of this work was to define the cellular function of TRPM6 and TRPM7 in cells either expressing only TRPM7 or co-expressing both proteins, TRPM6 and TRPM7. In the first line of our experiments, we generated and functionally characterized three different cell lines deficient in TRPM7 such as mouse trophoblast stem (TS) cells, human haploid leukaemia (HAP1) cells and primary isolated mouse megakaryocytes (MKs). Using the patch-clamp approach, we showed that all three cell lines lacked endogenous TRPM7 divalent cation-selective currents. We also found that TRPM7 deficient TS and HAP1 cells developed Mg2+ deficiency and growth arrest, which could be rescued by increased levels of Mg2+ in the culture medium. TRPM7 deficient MKs were viable but exhibited reduced Mg2+ contents and impaired proplatelet formation. Similar to TS and HAP1 cells, the changes in MKs were reversed by Mg2+ supplementation. We concluded that the TRPM7 channel controls the cellular Mg2+ uptake necessary for the cell proliferation. To get insights into the cellular role of the native TRPM6 protein, we generated TRPM6 deficient TS cells. We observed that, in contrast to TRPM7 KO TS cells, TRPM6 deficient TS cells were able to proliferate in a medium not fortified by additional Mg2+. Furthermore, the currents in TRPM6 KO TS cells were reduced and more sensitive to cytosolic Mg•ATP compared to the WT TS cells. These findings are in line with the notion that endogenous TRPM6 functions as a subunit of heteromeric TRPM6/M7 channel complexes, where TRPM6 potentiate Mg2+ currents due to offset of the inhibitory effect of Mg•ATP. Our experiments with the endogenous TRPM6 and TRPM7 channels were further verified using recombinant TRPM6 and TRPM7 proteins overexpressed in HEK 293 cells. We observed that the recombinant TRPM6 and TRPM7 channels contribute differently to the functional characteristics of the heteromeric TRPM6/M7 channels mimicking the situation with TS cells lacking endogenous TRPM6. Most remarkably, we found that association of TRPM6 with TRPM7 results in large TRPM6/M7 currents insensitive to cytosolic levels of Mg•ATP. Taken together, we conclude that ubiquitously expressed TRPM7 is required for the cellular uptake of Mg2+ and that this function cannot be compensated by TRPM6. Association of TRPM6 in heteromeric TRPM6/M7 channel complexes allows to maintain a high rate of Mg2+ uptake in transporting epithelial cells.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 27. April 2018
• 1. Bericht­erstatter:in: Conradt, Barbara