Figure 4. Amino-acid substitution in TMC1 alters the leak current.

(A) TMC1 with 10 putative transmembrane domains. The six substituted amino acids are highlighted as colored balls in the predicted positions, and the deafness (dn) truncation is at the third extracellular loop between TM5 and TM6. (B) Diagram of the analysis of leak current in cultured Tmc1-knockout OHCs (P3 + 1 DIV) expressing modified TMC1 (TMC1*). (C) Representative traces showing the rescue of leak conductance in OHCs by control full-length TMC1 (TMC1_WT), deafness TMC1 (TMC1_dn), TMC1-G411C (G411C), TMC1-M412C (M412C), TMC1-N447C (N447C), TMC1-D528C (D528C), TMC1-T532C (T532C), and TMC1-D569C (D569C). Perfusion contents are indicated below. An 800 nm step deflection was applied to the hair bundle every 10 s by a glass probe. The glass probe induced MET currents are marked ‘+', accompanying unwanted MET currents and electrical artefacts induced by switching the perfusion system (#). Note that the MET current was truncated to better show the leak current. (D) Quantification of rescue by mTMC1 constructs. I_Leak values: TMC1_WT, –49 ± 5 pA, G411C, –23 ± 3 pA; M412C, –40 ± 6 pA, N447C, –15 ± 1 pA; D528C, –22 ± 4 pA, T532C, –32 ± 4 pA, D569C, –23 ± 3 pA, TMC1_dn, –18 ± 3 pA. The rescue indexes of FL and dn were used to evaluate significant difference. Cell numbers are shown on each bar. The external solution contained 1.3 mM Ca²⁺. The holding potential was −70 mV. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ANOVA.

Figure 4—figure supplement 1. Cysteine substitution in TMC1 affects the MET current and the leak current.

(A) Plots of amplitude of the background current recorded from Tmc1-knockout OHCs expressing engineered TMCs as indicated, before and after MTSET treatment. (B) Representative trace of I_m recording in a P1 + 3 DIV Tmc1;Tmc2 double-knockout OHC expressing TMC1-M412C. A 10 Hz train of 800 nm step deflection was applied to the hair bundle by a glass probe. (C) Summary of absolute values and normalized ratios of I_Leak and I_MET. The I_Leak values were measured from data in Figure 4. The restored MET values of all TMC1 constructs were measured from Pan et al. (2018), excepting that of dn, which was collected in vestibular hair cells from Kawashima et al. (2011).