TRPML1 gating modulation by allosteric mutations and lipids.

Area of Science:

• Molecular biology
• Biochemistry
• Structural 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 modulated by various ligands, including lipids and synthetic molecules, through allosteric regulation.

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 phosphoinositides.

Main Methods:

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

Main Results:

• Tyr404 was identified as a critical residue for TRPML1 allosteric regulation.
• Mutations at Tyr404 resulted in gain-of-function (Trp) and loss-of-function (Ala) channels.
• The structure revealed PI(4,5)P2 binding at a site overlapping with agonist/antagonist hotspots, explaining lipid inhibition.
• An endogenous phospholipid, likely sphingomyelin, was found at the same site, explaining sphingomyelin's inhibitory effect.

Conclusions:

• Tyr404 mutations provide novel tools for studying TRPML1 allosteric modulation and for drug discovery.
• The structural insights into PI(4,5)P2 and sphingomyelin binding offer a mechanistic understanding of TRPML1 inhibition.
• These findings advance the understanding of TRPML1 channel function and its role in MLIV pathogenesis.