Fig. 4: Structural basis underlying functional changes in the I607T disease-causing mutant.
a, b Representative current recordings of ClC-3 expressed in H293T cells with endogenous proton sensitive PAC channel knocked out at extracellular pH 7.3 or pH 5.0. The voltage step protocol (ranging from −80 to +160 mV with a 20 mV step for 300 ms) was employed. c, d Representative current recordings of I607T mutant (based on the mClC-3bS3/S2/S1 background) expressed in H293T cells with endogenous proton sensitive PAC channel knocked out at extracellular pH 7.3 or pH 5.0. The voltage step protocol ranging from −80 to +160 mV (with a 20 mV step) for 300 ms was employed. e, f Overall cartoon structure of mClC-3I607T_apo or mClC-3apo was aligned to reveal clear conformational changes within the dimer interface. g Structural alignment of mClC-3I607T_ATP and mClC-3ATP revealed the substantially decreased size induced by the I607T mutation. h Packing of dimer interface in ClC-3 has been tightened by the I607T mutation. i Residues within the dimer interface of mClC-3I607T_ATP critical for dimer interface packing are shown in blue. j, k Representative current recordings of I607T-F598A double mutant (based on the mClC-3bS3/S2/S1 background) expressed in H293T cells with endogenous proton sensitive PAC channel knocked out at extracellular pH 7.3 or pH 5.0. The voltage step protocol was employed. l, m Representative current recordings of I607T-E599A double mutant (based on the mClC-3bS3/S2/S1 background) expressed in H293T cells with endogenous proton sensitive PAC channel knocked out at extracellular pH 7.3 or pH 5.0. The voltage step protocol was employed. n, o Average current-density voltage relationship of ClC-3 and mutants measured in H293T cells with endogenous proton sensitive PAC channel knocked out at extracellular pH 7.3 or pH 5.0. Mean ± SEM from biologically independent cells, n = 3. Two-way ANOVA, the exact P values were labeled. p Schematic model showing the proposed mechanism of ClC-3 potentiation by ATP. q Schematic model showing the proposed mechanism of I607T mutation induced functional changes in ClC-3.
