Characterize TMEM94 as ER Mg2+ uptake machinery and pathophysiological consequences.

ABSTRACT The intracellular Mg2+ (iMg2+) has essential roles as a counterion for negative charges, stabilizer of molecular and macromolecular structures, and cofactor for many enzymatic reactions in living cells. In eukaryotes, Mg2+ is the most abundant divalent cation essential for cellular functions and its perturbation is associated with several clinical conditions including migraines, obesity, diabetes, cardiovascular diseases, and preeclampsia however, the molecular events that directly link Mg2+ to cardiovascular disease progression are still elusive. Although the consensus is that free ionized intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids and proteins (17-30 mM), molecular mechanisms that control free iMg2+ (~0.5-1.2 mM) compartmentalization is limited. Fluctuations in free cytosolic Mg2+ (cMg2+) following stimulation have been touted as passive adjustments of Mg2+ dissociating from the exuberant Mg-ATP and other buffered pools. Our recent identification of small ligand-like activators opened a new avenue to understand the iMg2+ dynamics and the cause-effect relationships that exist between iMg2+ flux and cellular processes (Cell 2020). Having proved L-lactate as a ligand for ER Mg2+ (ERMg2+) dynamics, we asked whether the iMg2+ is compartmentalized and the existence of Mg2+ transporters across the subcellular membranes. Our recent discovery revealed that ER/SR is the primary site for iMg2+ store that is refilled by ER localized P-type Mg2+ ATPase (ERMA) (Molecular Cell 2024). Although ERMA is discovered as an ER localized P-type Mg2+ ATPase, it is unknown whether ERMA is localized in the SR membrane as an integral protein in cardiomyocytes. The highly conserved ERMA is exclusively localized on the ER/SR membrane with a Mg2+ binding signature motif. Remarkably, ERMA human mutations manifest neurodevelopmental disorders, congenital heart malformations, and facial dysmorphism. To explore the functional role of ERMA in cardiac cell types, we conducted the expression, localization and measured the activity of ERMA in adult cardiomyocytes. Our finding show that ER/SR is the primary reservoir for free Mg2+ ions in most cell types including cardiomyocytes. Based on this finding, we hypothesize that ERMA is a major Mg2+ transporting P-ATPase responsible for the uptake of cMg2+ into the ER/SR. Our three specific aims will:1. Establish ERMA-mediated SRMg2+ uptake in cardiomyocytes. 2. Determine the conserved domains, post- translational modification of ERMA that are crucial for SR/ERMg2+ uptake. Additionally, we will determine the structure function of ERMA and its oligomeric nature for the activity, and 3. Discover the physiology and pathogenesis associated with ERMA mutations in cardiac myocytes and cardiac function in vivo. The outcomes of this study will highlight potential new therapeutic targets for the treatment of conditions associated with cardiac diseases.