Surfe2r n1

Automated SSME recordings were performed on the SURFE²R N1 and the SURFE²R 96SE (Nanion Technologies), using the SURFE²R N1 Control 1.7.0.2 and the SURFControl96 1.7 software, respectively.
Technical details of both devices have been reviewed recently [33 (link),34 (link)] and are summarized in the Supplementary Materials. In brief, both instruments perform a fast solution exchange from non-activating solution to activating solution at 0 mV, applying a substrate gradient to activate charge translocation of TMEM175 in lysosomes, which are immobilized on a gold-coated sensor chip. The SURFE²R N1 is a single-well device, while the SURFE²R 96SE enables high-throughput sequencing by utilizing 96-well sensor plates, facilitating 96 simultaneous recordings. Although the devices employ different liquid handling methods and electrophysiological hardware, the measurement principle, experimental conditions, and workflows used are fundamentally identical.

SSME was performed using the SURFE²R N1 device (Nanion Technologies GmbH) and sensor preparation followed the standard protocols as described in detail previously (Bazzone et al., 2017a (link); Bazzone and Barthmes, 2020 (link)). A summary is provided in Supplementary Methods S1.1.
If not stated otherwise, all current traces shown in the same graph were recorded on the same sensor. For analysis, only the currents during the activating solution (A) flow were used. The time resolution of the solution exchange depends on the sensor surface area, with about 3–6 ms for 1 mm sensors and 20–40 ms for 3 mm sensors (Bazzone et al., 2017a (link)) and may limit the recording of fast PSS currents.
The buffer used to prepare the measurement solutions (R, A and non-activating solution, NA) contained 30 mM Tris/HCl, 3 mM EDTA, 1 mM EGTA and 120 mM NMDG/SO₄ at pH 7.4. Each figure includes a scheme describing the additional components of NA, A and R solutions used for the respective experiment. Details about the preparation of measurement solutions can be found in Supplementary Methods S1.2.

SSM electrophysiology experiments were performed as previously described (Bazzone et al., 2017 (link); Kermani et al., 2018 (link))using a SURFE²R N1 instrument (Nanion Technologies, Munich, Germany). The SSM sensors were prepared using 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in a non-activating buffer containing 100mM KCl, 100 mM KPO₄ pH 7.5. Proteoliposomes diluted 25-fold in buffer were sonicated and fused to the sensor lipid layers by centrifugation at 2500 g for 30 min. After both the lipidation and proteoliposome fusion steps, the sensor capacitance and conductance were determined using SURFE²R software protocols. Only sensors with a capacitance between 15 and 35 nF were used for experiments. For transport measurements, sensors were perfused with test substrate (2 mM) in 100 mM KCl, 100 mM KPO₄ pH 7.5 buffer, and capacitive currents were recorded. To compare transport currents measured using different sensors, currents were normalized to the peak current generated by the first perfusion of the positive reference substrate, Gdm⁺, measured on that sensor. Data was collected from three independent sensor preparations.