Figure 5

Plasma membrane expansion activity of TMEM16F mutants (A) Single HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na⁺; transient increase of extracellular [Ca²⁺] to 2 mM (at the time shown) was used to induce an increase in intracellular Ca²⁺. The change in membrane capacitance (∆C_m) was measured and the percentage change was calculated. Single recordings are displayed, mean values at 30 s, 45 s and 60 s after stimulation are shown (+/−SEM, n = 7). Upper red trace, control cells; lower blue trace, cells over-expressing murine TMEM16F. (B) Single TMEM16F-null HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na⁺; transient increase of extracellular [Ca²⁺] to 2 mM (at the time shown) was used to induce an increase in intracellular Ca²⁺. The change in membrane capacitance (∆C_m) was measured and the percentage change was calculated. Single recordings are displayed with mean values at 30 s, 45 s and 60 s after stimulation shown (+/−SEM, n = 7). Upper red trace, control cells; lower traces, cells over-expressing murine TMEM16F and mutants as shown. (C) Single TMEM16F-null HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na⁺; a transient increase of extracellular [Ca²⁺] to 2 mM was used to induce an increase in intracellular Ca²⁺ in the presence of the phosphatidylserine binding dye K7r. In the case of “WT rescue (iono)” cells were treated with 5 μM ionomycin instead. The total fluorescence of each cell was measured at 60 s using a confocal microscope, and the percentage change compared to untreated was calculated. Mean values are shown (+/−SEM, n = 7; **p < 0.01, ns, not significant, Student t-test).