ABSTRACT
The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.
Subject(s)
Cardiac Electrophysiology , High-Throughput Screening Assays , Membrane Potentials , Cations , LysosomesABSTRACT
Caring for pediatric patients with a ventricular assist device (VAD) requires a collaborative approach from an interprofessional team to ensure maximum patient safety and optimal outcomes. Initiating a VAD program is challenging, due to the complex medical and technical nature of this device and associated learning needs. At our institution, the development of the interprofessional VAD support team was established in four phases. Initial Education, Core Team Formation, Expansion, and Evaluation. A "core VAD team" was created after the initial education at an established VAD center. In a third step, all efforts were directed toward increasing the number of health care professionals caring for the VAD patients in the Cardiac Critical Care setting and on the Cardiac ward. The last phase consists of ongoing evaluation. Several key areas imperative to the care of a patient on a VAD were identified and further elaborated. The complex care of a patient on a VAD needs a specialized team approach to cover all patient care needs. Ongoing interprofessional education continues to improve competency of care. Continuity of care was assured on all levels of service to ensure the best possible outcomes.
