50 mM TEA⁺ or TMA⁺ fail to block wild-type ELIC and a wider-pore mutant. (A) Macroscopic current responses to 5-s pulses of 10 mM cysteamine recorded from the L9′A + F16′L wider-pore mutant at –80 mV in the outside-out configuration. The responses were recorded without TEA⁺ in either barrel or in the presence of 50 mM TEA⁺ flowing through both barrels of the theta-type perfusion tubing. Because ELIC displayed marked rundown in outside-out patches, these traces were not recorded sequentially. Instead, individual patches of membrane were exposed to cysteamine pulses either in the absence or the presence of TEA⁺. The solutions flowing through the two barrels of the perfusion tubing were (in mM) 142 KCl, 5.4 NaCl, 1.8 CaCl₂, 1.7 MgCl₂, and 10 HEPES/KOH, pH 7.4, with or without cysteamine and with or without TEA⁺. In the schematic representations of the theta-tubing perfusion, arrows indicate the application of TEA⁺. (B) Peak-current amplitudes recorded in response to the application of cysteamine in the presence of external 5 or 50 mM TEA⁺ normalized to the peak value observed in the absence of TEA⁺. The plotted values are means obtained from 4 (5 mM TEA⁺) and 7 (50 mM TEA⁺) patches; error bars are the corresponding standard errors. (C and D) Macroscopic current responses to 5-s pulses of 10 mM cysteamine in the presence of external 50 mM TMA⁺ recorded from the wild type and the L9′A + F16′L mutant at –80 mV in the outside-out configuration. All other conditions are as in A, replacing TEA⁺ with TMA⁺. The corresponding responses in the absence of TMA⁺ are shown in Fig. 5 A (for the wild type) and in panel A of this figure (for the mutant). (E) Peak-current amplitudes recorded in response to the application of cysteamine in the presence of external 50 mM TMA⁺ normalized to the peak value observed in the absence of TMA⁺. The plotted values are means obtained from 9 (wild-type ELIC) and 8 (L9′A + F16′L mutant) patches; error bars are the corresponding standard errors.