Figure 6.

Effect of single amino acid mutations on whole-cell current and anion selectivity. (A) Mean peak I_Ca,Cl current density when activated by 250 µM free [Ca²⁺]_i of mock, mTMEM16F, and single amino acid mutants in the putative pore site of mTMEM16F (the control mutation I342A and the pore region mutants R592E, K616E, and R636E) measured at 70 mV and −110 mV, respectively. Data are presented as mean ± SE; you can read the amount of replicates inside the 110-mV bar graphs; “*” and “†” indicate significant (P < 0.05, when tested with one-way ANOVA) difference from mock and mTMEM16F, respectively. (B–D) Anion permeabilities recorded in whole-cell mode after simulation with 250 µM free [Ca²⁺]_i for I342A (B)-, K616E (C)-, and R636E-transfected (D) HEK293 cells. Data are presented as mean ± SE; the number of replicates are indicated in the figures; “*” indicates significant (P < 0.05) difference from PCl, and “†” indicates significant difference (P < 0.05) between the relevant anion permeabilities, when tested with paired t test. Only experiments in which the anion permeabilities were paired were included in the statistical test. (E) Putative topology of the TMEM16F channel with the mutated amino acids’ approximate positions. (F) Immunofluorescence images of localization of TMEM16F mutants expressed in HEK293 cells. The images were taken using an epifluorescence microscope. GFP was enhanced by anti-GFP and Alexa Fluor 488 (green). (G) Presence of mTMEM16F-GFP in the plasma membrane fraction and total cell lysate of HEK293 cells expressing either GFP-tagged WT mTMEM16F or one of the mutants I342A, R592E, K616E, and R636E. Plasma membrane proteins were isolated using a biotinylation assay. β-Actin and NPTII were used as controls for loading and transfection efficiency, respectively.