Agonist Modulation of TRPML3 Channel Gating

Mucolipidosis Type IV (MLIV) is an autosomal recessive lysosomal storage disorder (LSD) that results from loss of Transient Receptor Potential Mucolipin 1 (TRPML1) ion channel function. MLIV causes Alzheimer\u27s-like neurodegeneration within the first year of life with currently no FDA-approved therapies. The unmet medical need represents an opportunity to develop novel therapeutics based on a better understanding of TRPML ion channel function and structure. Synthetic small-molecule TRPML3 channel agonists that have been identified using electrophysiological approaches have been shown to activate disease-causing mutant TRPML1 channels isolated from MLIV patients. Here we first demonstrate that TRPML3 channel activity can be reliably measured using both automated high-throughput and conventional whole-cell voltage clamp methods. We also show that extracellular application of the synthetic agonist ML-SA1 and intracellular application of diC8-PI(3)P results in synergistic activation of TRPML3. Furthermore, we find that the TRPML3 gain-of-function mutant (A419P) produces constitutive currents that are insensitive to ML-SA1. Using novel TRPML3 atomic resolution structure in complex with native membrane lipids and solved by cryo-electron microscopy (cryo-EM) by our collaborator (Youzhong Guo, VCU School of Pharmacy), we identified a residue (R414) in S5 that is hypothesized to contribute to the formation of a potentially novel PI(3)P or PI(5)P binding site. We show that a neutralizing mutation (R414A) reduces the effects of ML-SA1 to activate TRPML3 channels. In summary, our studies contribute new knowledge about the structural basis of gating in WT and mutant TRPML ion channels that may lead to the development of novel treatments for a neurodegenerative disease