Molecular underpinning of intracellular pH regulation on TMEM16F

Abstract

TMEM16F, a dual-function phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection, and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pHi). We found that low pHi attenuates, whereas high pHi potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca2+ ([Ca2+]i). We further demonstrate that TMEM16F pHi sensitivity depends on [Ca2+]i and exhibits a bell-shaped relationship with [Ca2+]i: TMEM16F channel activation becomes increasingly pHi sensitive from resting [Ca2+]i to micromolar [Ca2+]i, but when [Ca2+]i increases beyond 15 µM, pHi sensitivity gradually diminishes. The mutation of a Ca2+-binding residue that markedly reduces TMEM16F Ca2+ sensitivity (E667Q) maintains the bell-shaped relationship between pHi sensitivity and Ca2+ but causes a dramatic shift of the peak [Ca2+]i from 15 µM to 3 mM. Our biophysical characterizations thus pinpoint that the pHi regulatory effects on TMEM16F stem from the competition between Ca2+ and protons for the primary Ca2+-binding residues in the pore. Within the physiological [Ca2+]i range, the protonation state of the primary Ca2+-binding sites influences Ca2+ binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pHi and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pHi, including cancer.

Figure 1.

pH_i regulates TMEM16F ion channel activity. (A) Representative TMEM16F currents recorded from inside-out patches perfused with intracellular solutions containing 100 µM Ca²⁺ at different pH_i values. Currents were evoked by voltage steps from −100 to +100 mV with 20 mV increments, and the holding potential was −60 mV. All traces shown were from the same patch. (B) Mean G-V relations of the TMEM16F channels under different pH_i values at 100 µM Ca²⁺. Relative conductance was determined by measuring the amplitude of tail currents 400 µs after repolarization to a fixed membrane potential (−60 mV). The smooth curves represent Boltzmann fits G/G_max = 1/{1 + exp[−ze(V − V_1/2]/kT}. G_max is tail current amplitude in response to depolarization to +100 mV in 100 µM Ca²⁺ at pH_i 8.9. Error bar represents SEM (n = 7). (C) G-pH_i relationship for TMEM16F channels. Data were normalized to pH_i 8.9. Dashed line represents Boltzmann fit. G-pH_i curves from 6.1 to 8.9 were fitted with linear regression shown as the solid lines, which aligned well with the Boltzmann fits (dashed line). Thus, the mean slopes from linear regression were used as a parameter for pH_i sensitivity in later experiments (n = 7).

Figure S1.

pH_i regulates TMEM16A ion channel activity. (A) Representative TMEM16A currents recorded from inside-out patches perfused with intracellular solutions containing 0.5 µM Ca²⁺ at different pH_i values. Currents were elicited by voltage steps from −100 to +100 mV with 20-mV increments. The holding potential was −60 mV. All the traces shown were from the same patch. (B) Mean G-V relations of the TMEM16A channels under different pH_i values at 0.5 µM Ca²⁺. Relative conductance was determined by measuring the amplitude of tail currents 400 µs after repolarization to a fixed membrane potential (−60 mV). The smooth curves represent Boltzmann fits: G/G_max = 1/{1 + exp[−ze(V − V_1/2)/kT]}. G_max is tail current amplitude in response to depolarization to +100 mV in 0.5 µM Ca²⁺ at pH_i 8.9. Error bar represents SEM (n = 7). (C) Mean conductance of TMEM16A at different pH_i values was normalized to the maximum conductance at pH_i 8.9 at different voltages and then plotted as a function of pH_i (G-pH_i relationship). Data were fitted with linear regression curves, and the mean slopes from fits were 0.27, 0.28, 0.29, 0.3, and 0.29 for 20, 40, 60, 80, and 100 mV, respectively (n = 7).

Figure S2.

Rundown of TMEM16F at different pH_i values using voltage-step protocol. (A) Representative TMEM16F currents recorded from inside-out patches perfused with intracellular solutions containing 100 µM Ca²⁺ at different pH_i values. The interval between each trace was 25 s, and all the recordings were from the same patch. (B) Normalized conductance as different time points shown in A. Error bars represent mean ± SEM (n = 4).

Figure S3.

Q559K, a pore lining residue mutation that eliminates channel rundown, has the same pH_i sensitivity as WT TMEM16F. (A) Representative TMEM16F-Q559K currents recorded from inside-out patches perfused with intracellular solutions containing 100 µM Ca²⁺ at different pH_i values. (B) The G-V curves of Q559K currents at different pH_i values. Error bars represent SEM (n = 5). (C) The pH_i sensitivity of Q559K (QK) evaluated by the G-pH_i relationship. The slope of the G-pH_i relationship for Q559K at 100 µM Ca²⁺ is 0.20 ± 0.02, shown as red solid line. The G-pH_i curves of WT at different Ca²⁺ concentrations are also plotted as dashed lines for reference.

Figure 2.

pH_i regulates TMEM16F lipid scrambling activity. (A) Schematic design of the lipid scrambling fluorometry assay. CaPLSase activity is monitored by cell-surface accumulation of fluorescently tagged AnV, a PS binding protein that is continuously perfused through a perfusion manifold. AnV fluorescence remains dim in bulk solution and will strongly fluoresce after being recruited by cell surface PS, which is externalized by CaPLSases. We use whole-cell patch pipettes to deliver intracellular solutions into the cytosol to achieve precise control of pH_i and Ca²⁺. Once breaking into whole-cell configuration, the pipette solution rapidly diffuses into the cell and activates CaPLSases. AnV fluorescence signal on the cell surface was continuously recorded with 5-s intervals. (B) Representative lipid scrambling fluorometry images of HEK293T cells stably expressed with TMEM16F-eGFP (left, green signal) at different pH_i values. For the AnV-CF 594 signal on the right, the first column is fluorescence signal immediately after forming whole-cell configuration; the second column is the time when fluorescence intensity reached half maximum (t_1/2), and the last column is the time when fluorescence signal reached roughly plateau. The time points (minutes followed by seconds) of each image after breaking into whole-cell configuration are shown on the top right corner. The pipette solution contained 100 µM Ca²⁺, and holding potential was −60 mV. See also Video 1. (C) The time course of AnV fluorescence signal at different pH_i values as shown in B. The AnV signal was normalized to the maximum AnV fluorescence intensity at the end of each recording. The smooth curves represent fits to generalized logistic equation, F = F_max/{1 + exp[−k(t − t_1/2)]}. (D) Under 100 µM Ca²⁺, the onset times (t_on), when AnV signal can be reliably detected, for pH_i 6.1, 7.3, and 8.9 are 18.3 ± 1.3 (n = 5), 11.3 ± 1.4 (n = 5), and 4.5 ± 0.7 min (n = 5), respectively. (E) Under 100 µM Ca²⁺, t_1/2 values for pH_i 6.1, 7.3, and 8.9 are 29.07 ± 1.35, 20.28 ± 1.51, and 11.59 ± 1.56 min (n = 5), respectively. (F) Under 100 µM Ca²⁺, slopes for pH_i 6.1, 7.3, and 8.9 are 0.19 ± 0.01, 0.26 ± 0.02, and 0.40 ± 0.02 (n = 5), respectively. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, using one-way ANOVA followed by Tukey’s test.

Figure S4.

TMEM16F KO HEK293T cells show no scrambling activity at all pH_i values tested. Representative fluorescence intensity of AnV binding for TMEM16F-eGFP stable HEK293T and TMEM16F-KO HEK293T cells at different pH_i values (n = 4 for pH_i 8.9; n = 3 for pH_i 6.1 and 7.3). See also Video 2.

Figure 3.

pH_i regulation of TMEM16F channel activity is Ca²⁺ dependent. (A and B) Representative TMEM16F currents recorded from inside-out patches perfused with intracellular solutions containing 5 and 1,000 µM Ca²⁺, respectively. All traces shown in each panel were from the same patch. Currents were elicited by voltage steps from −100 to +100 mV with 20-mV increments. The holding potential was −60 mV. (C and D) Mean G-V relations of the TMEM16F currents from A and B, respectively. Relative conductance was determined by measuring the amplitude of tail currents 400 µs after repolarization to a fixed membrane potential (−60 mV). The smooth curves represent Boltzmann fits. Error bars represent SEM (n = 5). (E) pH_i sensitivity of TMEM16F current at +100 mV was measured by the slope of the the G-pH_i relationship. Mean conductance at different Ca²⁺ concentrations was normalized to the maximum conductance at pH_i 8.9 and +100 mV. Averaged slopes from linear fit for 5 and 1,000 µM Ca²⁺ were 0.32 and 0.04, respectively. The G-pH_i curve at 100 µM Ca²⁺ was replotted as black line for reference. Error bars represent SD (n = 5).

Figure S5.

TMEM16A-CaCC loses pH_i regulation under saturating Ca²⁺. (A) Representative TMEM16A currents recorded from inside-out patches perfused with intracellular solutions containing 100 µM Ca²⁺ at different pH_i values. (B) G-V relationships at different pH_i values under 100 µM Ca²⁺. All conductances were normalized to the maximum conductance at pH_i 8.9 and +100 mV. Error bar represents SEM (n = 5). (C) G-pH_i curve of TMEM16A at 100 µM Ca²⁺ (red solid line) and 0.5 µM Ca²⁺ (black dotted line). The slopes from linear fits are 0.02 and 0.3, respectively. Error bar represents SEM (n = 5).

Figure S6.

pH_i has no effect on the gain-of-function TMEM16A and TMEM16F mutations when Ca²⁺ is absent. (A) Locations of L543 and Q645 on the TMEM16A structure (PDB 5OYB). The residue numbers correspond to TMEM16A (a). For TMEM16A (ac) splice variant, the residue numbers are L547 and Q649, respectively. (B) Representative TMEM16A-L543Q and TMEM16A-Q645A currents recorded from inside-out patches perfused with intracellular solutions containing 0 Ca²⁺ at different pH_i values. (C) I-pH_i curve of TMEM16A mutations L543Q and Q645A at 100 mV. Slopes from linear fit for L543Q and Q645A are −0.02 and −0.04, respectively. The G-pH_i curve of WT under 0.5 µM Ca² was replotted as the black dashed line. Error bars represent SEM (n = 5). (D) Locations of Y563 and F518 on the TMEM16F structure (PDB 6QP6). (E) Representative TMEM16F-Y563K and TMEM16F-F518K currents recorded from inside-out patches perfused with intracellular solutions containing 0 Ca²⁺ at different pH_i values (F) The I-pH_i relationship of TMEM16F mutations Y563K and F518K at 100 mV. Slopes from linear fit for Y563K and F518K are 0.03 and 0.01, respectively. G-pH_i curve of WT under 100 µM Ca²⁺ was replotted as black dashed line. Error bars represent SEM (n = 5). Note that the gain-of-function mutations do not have obvious tail current under 0 Ca²⁺; therefore, I-pH_i relations not G-pH_i were plotted to evaluate their pH_i sensitivities.

Figure 4.

pH_i regulation of TMEM16F scrambling activity is Ca²⁺ dependent. (A) Representative images of TMEM16F-eGFP scrambling activity under 5 µM intracellular Ca²⁺ with different pH_i values. The white dotted rectangles in the top row demarcate the patch-clamped TMEM16F-eGFP–expressing cells. For the AnV-CF 594 signals on the right, the first column is fluorescence signal immediately after forming whole-cell configuration; the second column is the time point (minutes followed by seconds, top right corner) when fluorescence intensity reaches half maximum (t_1/2), and the last column is the time point when fluorescence reaches plateau. No obvious AnV-CF 594 signal can be detected in pH_i 6.1 over 40 min. The recording interval is 10 s. See also Video 3. (B) The time courses of AnV fluorescence intensity for TMEM16F activated by 5 µM Ca²⁺ under different pH_i values in A. The smooth curves represent fits to the logistic equation similar to Fig. 2 B. (C) The scrambling onset time (t_on) at different pH_i values under 5 µM Ca²⁺. N/S at pH_i = 6.1 represents no scrambling. Error bars represent SEM (n = 5). (D) The t_1/2 at different pH_i values under 5 µM Ca²⁺. N/S at pH_i = 6.1 represents no scrambling. Error bars represent SEM (n = 5). (E) The slopes at different pH_i values under 5 µM Ca²⁺. (F) Representative images of TMEM16F-eGFP scrambling activity under 1,000 µM intracellular Ca²⁺ with different pH_i. For the AnV-CF 594 signal on the right, the first column is fluorescence signal immediately after forming whole-cell configuration; the second column is the time point when fluorescence intensity reaches half maximum (t_1/2), and the last column is the time point when fluorescence reaches plateau. See also Video 4. (G) Time courses of AnV fluorescence intensity for TMEM16F activated by 1,000 µM Ca²⁺ under diff,erent pH_i values in F. The smooth curves represent fits to the logistic equation. (H) The scrambling onset time (t_on) at different pH_i values under 1,000 µM Ca²⁺. Error bars represent SEM (n = 5). (I) The t_1/2 of lipid scrambling at different pH_i values under 1,000 µM Ca²⁺. (J) The slopes (k) at different pH_i values under 1,000 µM Ca²⁺. Error bars represent SEM (n = 5). Statistics were analyzed using one-way ANOVA followed by Tukey’s test. **, P < 0.01; ***, P < 0.001; ns (not significant), P > 0.05.

Figure 5.

pH_i alters Ca²⁺ binding affinity of TMEM16F. (A) Representative TMEM16F currents recorded from inside-out patches perfused with intracellular solutions containing 0.1, 1, 5, 100, 1,000, and 5,000 µM Ca²⁺ at different pH_i values (5,000 µM at pH 6.1 only). (B) Ca²⁺ dose–response of mTMEM16F channel at +100 mV with different pH_i values. The smooth curves represent the fits to the Hill equation: G/G_max = G_1,000/[1 + (K_d/[Ca²⁺])H], where K_d is the apparent dissociation constant, H is the Hill coefficient, and G_1,000 is the conductance with 1,000 µM Ca²⁺ at given pH_i. The error bars represent SEM (n = 5). (C) EC₅₀ values of Ca²⁺ at pH_i 6.1, 7.3 and 8.9 were 144.45 ± 6.80, 6.20 ± 0.82, and 1.24 ± 0.14 µM, respectively. The error bars represent SEM (n = 5). P values were determined with Tukey test comparisons after one-way ANOVA: ****, P < 0.0001. (D) The G-pH_i relationship of TMEM16F current at +100 mV under different Ca²⁺ concentrations. Solid lines represent linear fits. (E) The relationship of pH_i sensitivity and [Ca²⁺]_i concentration. The pH_i sensitivity values were slopes from linear fit shown in D under different Ca²⁺ concentrations. The smooth line was fitted with a bell-shaped dose–response curve using GraphPad Prism, with peak pH sensitivity of 0.33 at ∼15 µM Ca²⁺. The error bars represent SEM (n = 5).

Figure 6.

Ca²⁺ binding sites mediate pH_i regulation on TMEM16F. (A) Representative TMEM16F-E667Q currents recorded from inside-out patches perfused with intracellular solutions containing 0.1, 1, 5, 10, and 20 mM Ca²⁺ at different pH_i values. (B) Ca²⁺ dose–response of TMEM16F-E667Q mutation. The error bars represent SEM (n = 4). (C) EC₅₀ values of Ca²⁺ at pH_i 6.1, 7.3, and 8.9 were 8.64 ± 1.08, 2.93 ± 0.42, and 1.22 ± 0.31 mM, respectively. The error bars represent SEM (n = 4). P values were determined with Tukey test comparisons after one-way ANOVA: ***, P < 0.001; *, P < 0.5. (D) The G-pH_i relationship of TMEM16F-E667Q. Solid lines represent linear fits. (E) The pH_i sensitivity and [Ca²⁺]_i concentration relationship of E667Q (solid line). The peak pH sensitivity is 0.22 at ∼3.01 mM Ca²⁺. The error bars represent SEM (n = 4). Curve from WT (dashed line) was replotted here for reference.

Figure S7.

Mutations of Ca²⁺ site near the dimer interface do not alter pH_i regulation on TMEM16F. (A and B) Representative TMEM16F-D859A and TMEM16F-E395A currents recorded from inside-out patches perfused with intracellular solutions containing 100 µM Ca²⁺ at different pH_i values. (C and D) The G-V curves of D859A and E395A currents, respectively. Error bars represent SEM (n = 5). (E) The pH_i sensitivity of D859A (red) and E395A (blue) evaluated by the G-pH_i relationship. The G-pH_i curves of WT at different Ca²⁺ concentrations were also plotted as dashed lines (black) for reference.

Figure 7.

Schematic model of pH_i regulation on TMEM16F and TMEM16A under physiological Ca²⁺_i. Under low pH_i conditions, protonation of the primary Ca²⁺ binding residues in the pore can significantly reduce Ca²⁺ binding affinity of TMEM16 proteins and suppress their activation. In contrast, high pH_i deprotonates the Ca²⁺ binding residues and enhances Ca²⁺ binding and TMEM16 activation. The size of blue arrows represents the open probability of TMEM16 proteins. The size of Ca²⁺ ions represents the strength of apparent Ca²⁺ binding affinity.