TMEM16A, A Membrane Protein Associated with Calcium-Dependent Chloride Channel Activity
Antonella Caputo1,
Emanuela Caci1,
Loretta Ferrera1,
Nicoletta Pedemonte2,
Cristina Barsanti1,
Elvira Sondo1,
Ulrich Pfeffer3,
Roberto Ravazzolo1,
Olga Zegarra-Moran1, and
Luis J. V. Galietta1,2*
1 Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Genova 16148, Italy.
2 Centro di Biotecnologie Avanzate, Genova 16132, Italy.
3 National Cancer Research Institute, Genova 16132, Italy.
|
Fig. 1.. Down-regulation of Ca2+-dependent anion transport by TMEM16A silencing. (A to C) CaCC assay based on the halide-sensitive YFP in pancreatic CFPAC-1 cells. (A) Representative traces showing cell fluorescence quenching upon the addition of extracellular I– plus UTP (arrow). (B) Summary of Ca2+-activated I– influx in untreated cells, in cells transfected with non-targeting siRNA (NT-siRNA), or transfected with siRNA pools against the indicated genes (mean ± SEM, n = 8 experiments per condition). (C) Ca2+-activated I– influx as in (B) but with a single siRNA against TMEM16A versus three nontargeting siRNA with different guanosine and cytosine content (mean ± SEM, n = 5 experiments per condition). Cells were stimulated with UTP (100 µM) or ionomycin (1 µM). ** P < 0.01 versus nontargeting siRNA. (D and E) Short-circuit current (Isc) recordings on polarized monolayers of primary human bronchial epithelial cells. Ca2+-dependent Cl– secretion was triggered with apical UTP (100 µM). Cells were transfected with nontargeting or anti-TMEM16A siRNA and then left in control conditions (D) or incubated with IL-4 (E). (Top) Representative traces. (Bottom) Summary of results (mean ± SEM, n = 4 experiments per condition). ** P < 0.01 versus nontargeting siRNA. (F) Whole-cell membrane currents (Im) from CFPAC-1 cells. Pipette (intracellular) solution contained 600 nM free Ca2+. (Top) Representative currents elicited at voltages in the –100- to +100-mV range. (Bottom) Membrane currents measured at the end of voltage pulses (Vm) (mean ± SEM, n = 10 to 20 experiments) are plotted against the applied membrane potential. Conditions were nontransfected, transfected with nontargeting siRNA, transfected with anti-TMEM16A, and nontransfected but with nominal 0 Ca2+ in the intracellular solution. The currents measured in TMEM16A-silenced cells were significantly smaller than those of control-transfected cells at +20 mV (P < 0.05) and at +40 to +100 mV (P < 0.01).
[View Larger Version of this Image (25K GIF file)]
|
|
|
Fig. 2.. CaCC activity in stable-transfected FRT cells. (A) Short-circuit current recordings on FRT cells. (Left) Representative experiments showing the effect of apical ionomycin addition (arrows) in null cells, a pool of cells transfected with TMEM16A, or a pure clone (no. 77) with stable expression of TMEM16A(abc). (Right) Summary of results (mean ± SEM, n = 4 to 5 experiments). Currents on clone 77 were also measured in the presence of niflumic acid (100 µM). ** P < 0.01 versus null cells. (B) Immunofluorescence images from FRT cells stably-transfected with the FLAG-tagged TMEM16A(abc) (top) and from null FRT cells (bottom). Cells were stained with antibody to FLAG (green) and counterstained with 4',6'-diamidino-2-phenylindole (blue). (C) Whole-cell membrane currents recorded in FRT cells stably-transfected with TMEM16A(abc) or in null cells at the indicated intracellular free-Ca2+ concentration. (D and E) Current-voltage relationships for TMEM16A(abc)-expressing cells showing dependence on intracellular free Ca2+ (D) or extracellular Cl– (E). In the latter type of experiments, intracellular Ca2+ was 600 nM.
[View Larger Version of this Image (21K GIF file)]
|
|
|
Fig. 3.. Induction of Cl– currents by transient TMEM16A expression. (A) Representative membrane currents measured in HEK-293 cells. Each panel is the overlap of currents elicited in a single cell at membrane potentials in the –100 to +100 mV range. Cells were transfected with null plasmids or with plasmids coding for (abcd) or (0) isoforms. (B) Current-voltage relationships from experiments as those shown in (A). Each point is the mean ± SEM of currents measured at the end of voltage pulses (n = 27 to 65 experiments). Values for (abcd) and (0) isoforms were significantly larger than those of mock-transfected cells at positive (P < 0.01) and negative (P < 0.05) membrane potentials). (Inset) Current-voltage relationship for a representative cell transfected with the (abcd) isoform before (no asterisk) and after (asterisk) replacement of extracellular Cl– with gluconate. (C) Membrane currents from a cell transfected with TMEM16A(abcd) carrying the R563A mutation. The arrow shows tail currents with a slower decay as compared with the wild-type protein in (A). (D) Time constant values ( ) (mean ± SEM, n = 3 experiments per condition) determined by fitting tail currents of the wild-type protein and the R563A mutant with a single exponential function. Tail currents were measured at –60 mV after stepping the membrane to the indicated voltages. * P < 0.05; ** P < 0.01. (E) Membrane currents from a cell transfected with the Q757A mutant. (F) Current-voltage relationship from experiments with the Q757A mutant before and after replacement of extracellular Cl– with gluconate (mean ± SEM, n = 6 experiments per condition).
[View Larger Version of this Image (32K GIF file)]
|
|
