The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17β-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion.

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The cellular resting membrane potential (Vm) not only determines electrical responsiveness of excitable cells but also plays pivotal roles in non-excitable cells, mediating membrane transport, cell-cycle progression, and tumorigenesis. Studying these processes requires estimation of Vm, ideally over long periods of time. Here, we introduce two ratiometric genetically encoded Vm indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting Vm between cell groups. We investigated the influence of ectopic expression of K+ channels and their disease-causing mutations involved in Andersen-Tawil (Kir2.1) and Temple-Baraitser (KV10.1) syndrome on median resting Vm of HEK293T cells. Real-time long-term monitoring of Vm changes allowed to estimate a 40–50 min latency from induction of transcription to functional Kir2.1 channels in HEK293T cells. The presented methodology is readily implemented with standard fluorescence microscopes and offers deeper insights into the role of the resting Vm in health and disease.