Table 5.
Comparison between TMEM175 assays and general technical limitations in SSME, whole-cell APC, and manual LPC.
| SSME | Whole-Cell APC | Lysosomal Patch-Clamp | |
|---|---|---|---|
| Experimental conditions | |||
| Driving force | Stimulus: concentration jumps; no direct voltage control, but voltage may be applied via uncouplers [55] | Stimulus: voltage steps; additional ionic gradients | Stimulus: voltage steps; additional ionic gradients |
| Solutions | Wide range of assay conditions, including pH gradients, extreme pH values and non-native ionic concentrations | Limited by the requirements of live cells; potentially requires chemicals to increase giga-seal stability, i.e., Fluoride, BSA; or channel blockers to increase specificity of the currents for the target protein | Potentially requires chemicals to increase giga-seal stability, i.e., fluoride, BSA, or channel blockers to increase specificity of the currents for the target protein |
| Target membrane | Lysosomes, non-treated, stored at −80 °C; impurities with plasma membrane vesicles | Plasma membrane of live cells | Freshly isolated lysosomes, chemically pre-treated using vacuolin-1 for enlargement |
| Protein orientation | Right-site-out | Inside-out | Right-site-out |
| Technical limitations | |||
| Read-out | Capacitive currents; peak represents initial translocation rate; potential pre-steady-state currents triggered by ion/substrate binding [40] | Steady-state currents; potential pre-steady-state currents triggered by voltage steps | Steady-state currents; potential pre-steady-state currents triggered by voltage steps |
| Signal-to-noise | Up to 1000-fold larger currents compared to whole-cell APC due to large circular sensor surface (Ø 3 mm) | Signal-to-noise is limited by surface of the cell and the expression of the target protein inside the target membrane | Signal-to-noise is limited by the surface of the lysosome and the expression of the target protein inside the target membrane |
| Signal interpretation | Signal represents flux of the ion species provided during fast solution exchange | Voltage steps stimulate the flux of all available ions across the membrane | Voltage steps stimulate the flux of all available ions across the membrane |
| Control experiments reveal… | Solution exchange artifacts due to membrane-ion interaction [41]; off-target compound effects using high compound concentrations | Off-target leak currents; off-target compound effects potentially smaller compared to SSME | Off-target leak currents; off-target compound effects |
| Ease of use | Easy, overall process is automated | Easy, overall process is automated | Highly skilled technician required |
| Throughput | 96 parallel recordings; lysosomes stored for months at −80 °C; sensor preparation in batches; stable sequential recordings | 384 parallel recordings; running cell culture required; duration of recordings limited by giga-seal stability | No parallel recordings; fresh lysosomal preparations each day; duration of recordings limited by giga-seal stability, further reduced compared to whole-cell APC |
| Data quality | Ultra-high success rates (96.4 ± 3.3%); superior z’ prime (0.87 ± 0.0278); low standard deviation between sensors | High success rates (82 ± 5.2%); superior z’ prime (0.768 ± 0.058); high standard deviations between cells | Low current amplitudes and lower success rates compared to APC and SSME; higher standard deviation between lysosomes |
| Novelty | Not yet standard in drug screening | Well-established standard method | Gold standard for lysosomal channels |