Figure 7. Gating of Heterologously Expressed mTMEM16F and Endogenous TMEM16F Channels in Megakaryocytes Is Ca²⁺ and Voltage Dependent.

(A) Representative macroscopic current traces of an inside-out patch excised from a mTMEM16F-expressing HEK293 cell exposed to 1, 2, 7, and 100 μM [Ca²⁺]_i. Testing potentials were from −80 mV to +180 mV with 20 mV increments. Both holding and repolarizing potentials were −80 mV.

(B) Mean G-V relations of the mTMEM16F channels under different [Ca²⁺]_i. Relative conductance was determined by measuring the amplitude of tail currents 400 μs after repolarization to a fixed membrane potential (−80 mV). The smooth curves represent Boltzmann fits I/I_Max = 1/(1 + exp(−ze(V−V_1/2)/kT)) (see Table S1). I_Max, tail current amplitude in response to deploarizaton to +180 mV in 100 μM [Ca²⁺]_i. Error bar represents SEM.

(C) Ca²⁺ dose-response of the mTMEM16F channel at +60, +120, and +180 mV. I/I_Max values were from (B). The smooth curves represent the fits to the Hill equation: I/I_Max = Amp/(1 + (K_D/[Ca])^H), wherein K_D is the apparent dissociation constant, H is the Hill coefficient, and Amp is the maximum value of I/I_Max at certain voltage. +60 mV: (K_D = 10.8 ± 4.6 μM, H = 2.8); +120 mV: (K_D = 5.4 ± 1.6 μM, H = 1.5); +180 mV: (K_D = 3.4 ± 0.5 μM, H = 1.8). n = 5–21. Error bar represents SEM.

(D) Representative inside-out patch-clamp recordings of WT (top) and E667Q mutant (bottom) mTMEM16F channels expressed in HEK293 cells. The membrane patches were voltage clamped at +60 mV and exposed to different [Ca²⁺]_i.

(E) Comparison of Ca²⁺ sensitivity between the WT and E667Q mutant TMEM16F channels at +60 mV. The smooth curves represent the fits to the Hill equation. WT: K_D = 13.6 ± 1.8 μM, H = 2.2 ± 0.5; E667Q: K_D = 2.8 ± 0.3 mM, H = 1.6 ± 0.3. n = 11–17. Error bar represents SEM.

(F) Representative inside-out patch-clamp recordings from the mTMEM16F-expressing HEK293 cells (top) and megakaryocytes of WT mice (bottom). The membrane patches were voltage clamped at +60 mV and exposed to different [Ca²⁺]_i.

(G) Comparison of the Ca²⁺ sensitivity of the endogenous TMEM16F-SCAN current in megakaryocytes with that of the heterologously expressed TMEM16F channels in HEK293 cells at +60 mV. The smooth curves represent the fits to the Hill equation. For mTMEM16F currents in HEK293 cells, K_D = 13.9 ± 2.9 μM, H = 1.6; for endogenous TMEM16F currents in megakaryocytes, K_D = 5.1 ± 2.0 μM, H = 2.5. n = 6–12. Error bar represents SEM.

All experiments were recordings from inside-out membrane patches in symmetrical 140 mM NaMES solutions except for the experiments shown in (D) and (E), which were done in symmetrical 140 mM NaCl solutions.

See also Figure S6 and Table S1.