Ion specificity of electrogenic behavior of KtrB analyzed using SSM-based electrophysiology. The results of the SSM-based experiments for the cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ are shown in green, orange, purple, red, and blue, respectively. (a) Transient currents induced by 100 mM concentration jumps of LiCl, NaCl, KCl, RbCl, and CsCl on one sensor. (b) Bar graph of the peak currents in panel a performed experiments normalized to the highest peak current (CsCl). All data are corrected by subtraction of empty liposome signals (±SEM, n = 3). (c) Bar graph of integrated transient currents (0.52–1 s) normalized to the biggest area (Na⁺). The normalized electrical (norm. electr.). charges were 0.25 ± 0.11 for Li⁺, 1 for Na⁺, 0.91 ± 0.09 for K⁺, 0.68 ± 0.16 for Rb⁺, and 0.44 ± 0.04 for Cs⁺. All data are corrected by subtraction of empty liposome signals (±SEM, n = 3). (d–g) Concentration jumps with increasing salt concentrations are indicated by rising color intensities (1–50 or 100 mM). All data were corrected by subtraction of empty liposome signals. For determining the K_m values with OriginPro2017, peak currents were normalized (NPC) to the highest concentration jump during one set of measurements (±SEM, n ≥ 3). The determined K_m values are 35.5 ± 1.9 mM for Na⁺ (d), 16.4 ± 1.8 mM for K⁺ (e), 5.9 ± 0.6 mM for Rb⁺ (f), and 2.1 ± 0.3 mM for Cs⁺ (g). (h) Concentration jumps with increasing KCl concentrations (1–100 mM) in the presence of 5 mM NaCl in the SSM buffer are shown. For K_m evaluation, peak currents were normalized (NPC) to the 100 mM concentration jump and fitted with OriginPro 2017.