TMEM16 proteins produce volume-regulated chloride currents that are reduced in mice lacking TMEM16A

Abstract

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca(2+)-activated Cl(-) channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y(2) receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca(2+) and a Ca(2+)-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl(-) conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of I(Cl,swell) was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl(-) channels and may also have a function during proliferation and apoptotic cell death.

FIGURE 1.

Activation of I_Cl,swell in CFPAC cells requires TMEM16A channels. A, RT-PCR analysis indicates expression of TMEM16A, TMEM16F, and TMEM16H in CFPAC cells. M, marker; A–K, TMEM16A–K. G_s, G_l, short and long splice variants. B, original recordings of whole cell currents in CFPAC cells activated by hypotonic bath solution (33%). Cells were voltage clamped in intervals from −50 to +50 mV. Treatment with siRNA for TMEM16A (si16A) reduced the swelling-activated whole cell current, when compared with cells treated with scrambled (scrbld) RNA. C, summary of swelling-activated whole cell conductance (G_Hypo) measured in CFPAC cells treated with scrambled RNA or after RNA interference knockdown of TMEM16A, CLC-3, and CLCA proteins. Mean ± S.E., (n) = number of cells measured. #, significant inhibition of I_Cl,swell by RNA interference knockdown of TMEM16A when compared with treatment with scrambled RNA (unpaired t test).

FIGURE 2.

Activation of I_Cl,swell in HT₂₉ cells requires TMEM16A channels. A, RT-PCR analysis indicates expression of TMEM16A, TMEM16F, TMEM16H, and TMEM16J in HT₂₉ cells. M, marker; A–K, TMEM16A-K; G_s, G_l, short and long splice variants. B, original recordings of whole cell currents in HT₂₉ cells, activated by a gradual increase of extracellular hypotonicity (17, 25, and 33%, arrows). Cells were voltage clamped in intervals from −50 to +50 mV. Partial replacement of extracellular Cl⁻ by gluconate (open bar, 30 mm Cl⁻) inhibited whole cell outward currents. Treatment with siRNA for TMEM16A (si16A) reduced the swelling-activated whole cell current, when compared with cells treated with scrambled (scrbld) RNA. C, summary of whole cell conductance measured in HT₂₉ cells under control conditions (open bars; normotonic bath solution) and after exposure to 33% hypotonicity (black bars). Mean ± S.E., (n) = number of cells measured. *, significant increase in whole cell conductance (paired t test). #, significant inhibition of I_Cl,swell by RNA interference knockdown of TMEM16A, when compared with treatment with scrambled RNA (unpaired t test).

FIGURE 3.

Activation of I_Cl,swell in HEK293 cells requires TMEM16A channels. A, RT-PCR analysis indicates expression of TMEM16A, TMEM16F, and TMEM16H in HEK293 cells. PCR product of TMEM16A becomes only visible when cDNA input was doubled. M, marker; A–K, TMEM16A–K; G_s, G_l, short and long splice variants. B, original recordings of whole cell currents in HEK293 cells, activated by a gradual increase of extracellular hypotonicity (17, 25, and 33%, arrows). Cells were voltage clamped in intervals from −50 to +50 mV. Partial replacement of extracellular Cl⁻ by gluconate (open bar, 30 mm Cl⁻) inhibited whole cell outward currents. Treatment with siRNA for TMEM16A (si16A) reduced the swelling-activated whole cell current, when compared with cells treated with scrambled (scrbld) RNA. C, summary of whole cell conductance measured in HEK293 cells under control conditions (open bars, normotonic bath solution) and after exposure to 33% hypotonicity (black bars). Mean ± S.E., (n) = number of cells measured. *, significant increase in whole cell conductance (paired t test). #, significant inhibition of I_Cl,swell by RNA interference knockdown of TMEM16A, when compared with treatment with scrambled RNA (unpaired t test).

FIGURE 4.

Activation of I_Cl,swell in HEK293 cells depends on the presence of TMEM16 channels. A, Western blot analysis indicates expression of endogenous TMEM16A in HEK293 cells. B, real time PCR analysis of expression of TMEM16A in HEK293 cells treated with scrambled siRNA (scrbld) and TMEM16A-siRNA (si16A). C, inhibition of swelling-activated whole cell conductance (G_Hypo) in HEK293 cells by DIDS (100 μm), CaCCinh-A01 (10 μm), and tamoxifen (100 μm). D, summary of G_Hypo in HEK293 cells expressing empty plasmid (mock), TMEM16A, or TMEM16B. E, summary of swelling-induced whole cell conductance in cells treated with siRNA for various TMEM16 proteins. Mean ± S.E., (n) = number of cells measured. #, significant difference when compared with control cells (mock, scrbld, or absence of inhibitors) (unpaired t test).

FIGURE 5.

Role of purinergic signaling for activation of TMEM16A in HEK293 cells by cell swelling. A, activation of whole cell conductance by increased extracellular hypotonicity in cells expressing empty plasmid (mock), TMEM16A or TMEM16A together with P2Y₂ receptors. B, swelling activation of whole cell currents in a HEK293 cell and inhibition of the current by suramin (100 μm; gray bar). C, inhibition of the swelling-activated whole cell conductance by suramin (100 μm), apyrase (3 units/ml), and ATP-free pipette solution, the cysteine reagent MTSET (2.5 mm), and the inhibitor of extracellular-regulated kinase (ERK1/2) U0126 (25 μm). D, summary of swelling-activated (Hypo) and ATP-induced whole cell conductance in TMEM16A expressing HEK293 cells, under control conditions and after removal of Ca²⁺ from both pipette and bath solution. Both ATP and swelling-induced whole cell conductance were reduced under Ca²⁺-free conditions and when two ERK1/2 consensus sides in TMEM16A were mutated. E, activation of whole cell currents by ATP (open bar, 10 μm), before and after swelling activation of whole cell currents (Hypo). F, summary of whole cell conductance in the absence and presence of ATP hypotonic bath solution. Mean ± S.E., (n) = number of cells measured. *, significant ATP effect (paired t test). #, significant difference when compared with control (unpaired t test). §, significant difference when compared with the absence of hypotonic bath solution (unpaired t test).

FIGURE 6.

TMEM16 proteins are essential for RVD in HEK293 cells. A, swelling (Hypo)-induced changes in calcein fluorescence in control HEK293 cells and cells treated with siRNA for TMEM16A or suramin (100 μm) or DIDS (100 μm); mean curve ± S.E. (n = 10). B, summary of the rate of fluorescence recovery per second, indicating recovery of the cell volume from hypotonic swelling. Treatment with siRNA for TMEM16A or exposure to various inhibitors of I_Cl,swell inhibited recovery of the cell volume, i.e. RVD. C, hypotonic cell swelling and recovery from cell swelling of cells treated with (i) scrambled RNA, (ii) TMEM16A-RNAi, or (iii) coexpressing P2Y₂ receptors/TMEM16A, as determined by FACS analysis of 30,000 cells per experiment. Experiments were performed in triplicate. D, summary of volume, i.e. fluorescence recovery after hypotonic cell swelling of HEK293 cells treated with siRNA for different TMEM16 proteins, as determined by FACS analysis of 30,000 cells per experiment. E, apoptotic cell shrinkage induced by staurosporin (2 μm) under normotonic conditions in cells treated with two different batches of siRNA for TMEM16A (si16A-a and si16A-b), or when treated with scrambled RNA (FACS analysis of 30,000 cells). Mean ± S.E., (n) = number of cells measured. *, significant recovery from cell swelling of cells treated with scrambled siRNA (paired t test). #, significant difference when compared with control (unpaired t test).

FIGURE 7.

Impaired I_Cl,swell in epithelial tissues of mice lacking TMEM16^−/−. A and B, whole cell currents and corresponding I/V curves obtained in submandibular acinar cells of TMEM16A knock-out mice (−/−) and wild type littermates (+/+) before and after exposure to hypotonic (33%) bath solution. C, summary of the whole cell conductance induced by hypotonic bath solution in WT and knock-out animals. D and E, whole cell currents and corresponding whole cell conductance obtained in hepatocytes of TMEM16A knock-out mice (−/−) and wild type littermates (+/+), before and after exposure to hypotonic bath solution. F, recording of the change in transepithelial voltage (V_te) induced by hypotonic bath solution (25%) in WT (+/+) and knock-out (−/−) animals. G, summary of ion transport, i.e. the short circuit currents induced by hypotonic bath solution in WT (+/+) animals and knock-out (−/−) littermates. Mean ± S.E., (n) = number of cells measured. *, significant increase in whole cell conductance (paired t test). #, significant difference when compared with WT (+/+) (unpaired t test).