Bi-allelic truncations in TMEM94 have been shown to cause congenital heart defects, facial dysmorphisms, and neurodevelopmental disorders, but the function of this membrane protein remains elusive. A study in 2024 proposed TMEM94 as a novel Mg2+ transporter located in the ER3, however, this study received criticisms that key experiments were missing to fully verify the mechanism of Mg2+ transport. In addition, the cryo-EM structure published this year lacks evidence supporting the function of TMEM94 as a Mg2+ transporter, potentially due to suboptimal experimental conditions used in the study. I am going to use structural, functional, and physiological studies to provide clarity to the role and mechanism of TMEM94 and its potential classification as a novel eukaryotic Mg2+ transporting P-type ATPase.

Aim 1: Determine the structure of TMEM94 in its active form. While recent cryo-EM studies have revealed the overall architecture of TMEM94, a substantial portion of the cytoplasmic region remains poorly resolved, likely due to the protein manipulation methods employed for purification. Since the cytoplasmic region is predicted to play a critical role in TMEM94’s function, the absence of high-resolution structural information in this domain significantly impedes mechanistic understanding. I aim to obtain the first high-resolution cryo-EM structure of TMEM94 in its active form, using conditions that preserve the integrity of the cytoplasmic region.

Aim 2: Assess whether TMEM94 functions as a primary active Mg2+ transporter. In parallel to the structural study, I aim to uncover the function of TMEM94 using in vitro reconstitution approach. Based on the published study by Vishnu et. al., my working hypothesis is that TMEM94 is a novel P-type ATPase that transports Mg2+. A critical part of the mechanism of P-type ATPases is the binding of ATP and hydrolysis to phosphorylate the P domain. This process moves the protein into the E2 conformation allowing ions to be released on the other side of the membrane. I plan to use Microscale Thermophoresis (MST) to test the binding of TMEM94 with ATP and Mg2+.