The biosynthetic anomaly of A531V is temperature-insensitive.

(A) Immunoblotting analyses of myc-tagged WT and A531V CLC-1 proteins from HEK293T cells incubated at 27°C for 48 hrs. (B) Quantification of total protein expression level by following the same procedure as described in Figure 4B. Data were obtained from 7 independent experiments. The mean normalized value of A531V is 0.70±0.09. Asterisks denote significant difference from WT (*, t-test: p(C) Averaged instantaneous I-V curves of the WT (black circles; n = 36) and the A531V mutant (red squares; n = 26) recorded under the cell-attached configuration. CLC-1 channel-expressing HEK293T cells were incubated at 27°C for 48 hrs. (D) Whole-cell current density (at −140 mV) of the A531V mutant recorded from HEK293T cells incubated at 37°C or 27°C.