Gramicidin a

For Single channel analysis (Figure 6), Gramicidin A (Sigma-Aldrich) was added into both aqueous phases surrounding a bilayer. The concentration of gA was 1 nM and the buffer solutions contained 1 M NaCl to enable electrophysiological measurements of the expected ion channels. After letting the system equilibrate for 15–20 min to form a stable bilayer, the bilayer was caught with a micropipette. To investigate the conductive properties of possibly formed pores, a silver chloride electrode connected to a patch-clamp setup was inserted into the micropipette (EPC10 – HEKA Germany). Using the single channel analysis mode, we recorded the current through the voltage-clamped membrane. Only after dispersing gA into the aqueous phases, an unitary amplitude of ≈2.8 pA is recorded, see Figure 6 (solid black line). This value is characteristic for gA pores under the used experimental conditions. As a control test, this experiment was repeated, yet adding 5 × 10^–3 M of calcium chloride (CaCl₂) into the buffer phases. The presence of divalent ions, such as calcium, is known to block the conductive properties of gA, when inserted in planar lipid bilayers. The presence of the divalent ion CaCl₂ blocked the conductive property of gA, inserted into a bilayer (as expected).

Wild‐type wing discs from up‐crawling third‐instar larvae were dissected in Grace's medium supplemented with 5% FBS and 20 nM 20‐hydroxyecdysone (full medium) (Dye et al, 2017) and incubated with 1 μM gramicidin A (Sigma‐Aldrich, #50845) in full medium for 30 min at 29°C. Before proceeding with either the membrane potential assay or PAC‐NAE uptake, wing discs were washed twice with full medium.

The following drugs were used: (−)Englerin A (EA, AppliChem, Darmstadt, Germany), clemizole hydrochloride (Clm, BioVision, Milpitas, CA), 4-methyl-2-(1-piperidinyl)-quinoline (ML204, Tocris Bioscience, Bristol, United Kingdom), ouabain (Sigma/Aldrich, St. Louis, MO), Pico145^{19 (link)}, and gramicidin-A (Sigma/Aldrich). Each drug except Pico145 was dissolved in the vehicle recommended by the manufacturer. Pico145 was dissolved in 100% DMSO to make 10 μM stoc^{19 (link)}.

GABA reversal potential recordings were made using a perforated patch-clamp technique. Pipettes were filled with an internal solution of 140 mM KCl and 10 mM HEPES (pH 7.3) containing gramicidin A 50 μg/ml (Sigma; diluted from a stock solution of 50 mg/ml in DMSO). Data were filtered during acquisition with a low pass filter set at 2 kHz using pClamp10 (Molecular Devices). Data analysis was performed offline with Clampfit 10.2 (Molecular Devices).

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) were bought from Avanti Polar Lipids. DOPC is used because it is one of the most common phospholipids present in human cells (Heo et al., 2019 (link)). The silicone oil (SiAR20) and squalene oil were purchased from Sigma-Aldrich. Ultra-pure water was obtained by filtration using a filtration system from Thermo Fisher. PDMS184 was purchased from Dow Corning. Gramicidin A and all other chemical, like salts: LiCl, NaCl, KCl, RbCl, CsCl, were purchased from Sigma-Aldrich. 0.1 M solute solution were prepared by dispersing the salt directly in pure water and under mechanical steering with 1 nM of gA.