DDX3X is a Cl ⁻ -sensitive RNA helicase

Cl^- homeostasis is pivotal during neurodevelopment and in multiple processes in mature neurons, and its disruption is implicated in several neurodevelopmental disorders. Here, we investigated the role of Cl^- in regulating DDX3X, an ATP-dependent RNA helicase that is associated with a neurodevelopmental disorder and is involved in stress granule assembly through biomolecular condensation. Cl^- directly interacted with the DDX3X helicase core in the RNA binding region. This interaction impaired both ATPase and RNA helicase activities at physiologically relevant concentrations in a manner similar to inorganic phosphate and disrupted its condensation propensity in vitro. In neuroblastoma cells, Cl^- efflux induced the formation of large, persistent DDX3X-containing stress granules. Furthermore, the R326H mutation, which is linked to a severe neurodevelopmental disorder, altered the chemical environment of the Cl^--binding site and impaired Cl^--sensitive functions. Together, our findings demonstrate that Cl^- binding regulates DDX3X functions and provide insights into the molecular pathophysiology of a neurodevelopmental disorder-linked mutation in DDX3X.