TRPML1 gating modulation by allosteric mutations and lipids.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel.
• Loss-of-function mutations in TRPML1 cause mucolipidosis type IV (MLIV), a lysosomal storage disorder.
• TRPML1 activity is allosterically regulated by various ligands, including lipids and synthetic molecules.

Purpose of the Study:

• To identify functionally critical residues in TRPML1 that mediate allosteric regulation.
• To characterize novel gain- and loss-of-function TRPML1 mutants as potential drug screening targets.
• To elucidate the structural basis of TRPML1 inhibition by PI(4,5)P2 and sphingomyelin.

Main Methods:

• Site-directed mutagenesis to create Tyr404 mutants (Trp, Ala).
• Functional characterization of mutant TRPML1 channels.
• High-resolution structural determination of TRPML1 in complex with PI(4,5)P2.

Main Results:

• Identified Tyr404 as a functionally critical residue; mutations Y404W and Y404A resulted in gain- and loss-of-function, respectively.
• Mutant channels mimic ligand effects without altering ligand binding and remain responsive to modulators.
• Determined the structure of TRPML1 with PI(4,5)P2, revealing the inhibition mechanism.
• Identified an endogenous sphingomyelin-like phospholipid at the agonist/antagonist binding site, explaining sphingomyelin's inhibitory effect.

Conclusions:

• Tyr404 is a key allosteric site for TRPML1 regulation, offering new avenues for therapeutic targeting in MLIV.
• The structural insights into PI(4,5)P2 and sphingomyelin inhibition provide a foundation for designing specific TRPML1 modulators.
• The developed gain- and loss-of-function mutants serve as valuable tools for high-throughput screening of TRPML1-targeting drugs.