Abstract P092: Palmitoylation Controls Cell Surface Abundance Of Trpm7

Abstract

Magnesium regulates numerous cellular functions and enzymes and abnormal magnesium homeostasis contributes to vascular dysfunction and the development of hypertension. The transient receptor potential melastatin 7 (TrpM7) has emerged as a key player in cardiovascular magnesium homeostasis. This bifunctional channel/kinase is ubiquitously expressed and regulates embryonic development. Its integral membrane ion channel domain regulates transmembrane movement of divalent cations, primarily Ca 2+ , Mg 2+ and Zn 2+ , and its kinase domain controls gene expression via histone phosphorylation. TrpM7 not only localizes on the cell surface to serve as a critical regulator of transmembrane Mg 2+ flux, but also forms an intracellular Zn 2+ release channel in vesicles of unknown origin. Palmitoylation is a dynamic reversible posttranslational modification, which regulates ion channel activity, stability, and subcellular localization. We found that TrpM7 is palmitoylated in multiple cell types. Here we sought to identify palmitoylated cysteines and the functional consequences of TrpM7 palmitoylation in HEK cells. Mutation of Cysteines 1143, 1144 and 1146 on TrpM7 (TrpM7-AAA) to alanines reduced its palmitoylation by 68.4±8% (n=13; P <0.05), identifying this cluster of cysteines as the principal sites of palmitoylation. Fluorescent microscopy indicated that TrpM7-AAA was retained in the endoplasmic reticulum (ER), but when the palmitoylated cysteines in TrpM7 were replaced with the analogous regions of TrpM2 (M7/M2) or TrpM5 (M7/M5), the chimaeric proteins could exit the ER despite being 80.6±13% (n=3; P <0.05) and 70.6±17% (n=3; P <0.05) less palmitoylated than wild type respectively. Using membrane-impermeable biotinylation reagents we found that palmitoylation alters the balance of distribution of TrpM7 between vesicular and cell surface pools. The proportion of M7/M2 and M7/M5 reaching the cell surface membrane was decreased by 69.0±14% (n=3; P <0.05) and 51.4±9% (n=3; P <0.05) respectively, compared to wild type. Therefore, inhibiting TrpM7 palmitoylation reduced its cell surface abundance. The impact of palmitoylation on TrpM7 channel activity and its contributions to hypertension and vascular pathologies need further investigation.