Direct patch-clamp recording reveals TRPML2-dependent cation conductance across pathogen-containing vacuole membranes

Transient receptor potential mucolipin 2 (TRPML2), a cation channel encoded by MCOLN2 and localized to endolysosomal compartments, has been implicated in restricting Salmonella enterica replication through regulation of the Salmonella-containing vacuole (SCV). However, direct electrophysiological evidence of TRPML2 activity on the SCV membrane has been lacking. Using S. enterica serovar Typhi as a model, we optimized infection and patch-clamp conditions to directly access SCVs in infected THP-1 cells and record vacuolar ion currents. SCVs from wild-type cells exhibited robust inward currents at negative potentials that were reduced in TRPML2-knockout cells and enhanced by TRPML2 agonists. Under Mg²⁺-permeant conditions, SCVs displayed TRPML2-dependent Mg²⁺ conductance, supporting a role for TRPML2 in regulating Mg²⁺ availability during S. Typhi infection. Ca²⁺-permeant recordings further showed agonist-responsive inward currents on SCVs, indicating that SCV-localized TRPML2 retains broader divalent-cation conductance. Basal currents were also better maintained on SCVs than on bacteria-negative vacuoles isolated from uninfected bystander cells within the same cultures. PI3P reduced current rundown, suggesting that the phosphoinositide environment of SCVs may help sustain TRPML2 activity. Together, these findings provide the first direct electrophysiological characterization of a pathogen-containing vacuole and establish a tractable platform for investigating host ion-channel function at the host-pathogen interface.