Figure 2. Ca²⁺ oscillations in goblet cells control KChIP3 function.

(A) Time course of Ca²⁺ responses (normalized FURA-2AM ratio) obtained in differentiated HT29-18N2 cells in resting conditions exposed to different extracellular buffers: 1.2 mM CaCl₂ (left), 0.5 mM EGTA (center), or 10 µM dandrolene (right) (n = 30, inset shows a recording obtained from a single cell under each condition). (B) Percentage of cells oscillating in each condition during 10 min. Average values ± SEM are plotted as scatter plot with bar graph (N > 3) (black dots: 1.2 mM CaCl₂, blue dots: EGTA, red dots: dandrolene). (C) Secreted MUC5AC collected from differentiated HT29-18N2 cells that were incubated for 30 min at 37°C with different buffers: 1.2 mM CaCl₂ (black dots), 0.5 mM EGTA (blue dots) or 10 µM dandrolene (red dots). The y-axis represents relative values with respect to the values of control cells. Average values ± SEM are plotted as scatter plot with bar graph (N > 3). (D) Secreted MUC5AC from differentiated control (black circles) and KChIP3 stable knockdown cells (KChIP3-KD) (blue squares) collected after 30 min incubation at 37°C in the in the presence (1.2 mM CaCl₂) or absence (0.5 mM EGTA) of extracellular Ca²⁺. Data were normalized to intracellular actin levels. The y-axis represents normalized values relative to the values of untreated control cells. (E) Secreted MUC5AC from control (black circles) and KChIP3 stable knockdown cells (KChIP3-KD) (blue squares) that were incubated for 30 min at 37°C with vehicle or 10 µM dandrolene (DAND) in the presence of extracellular Ca²⁺. Data were normalized to intracellular actin levels. The y-axis represents normalized values relative to the values of untreated control cells. (F) Secreted MUC5AC from differentiated control (black circles), KChIP3-GFP (red circles) and KChIP3-MUT (green circles) cells that were incubated for 30 min at 37°C in the in the presence (1.2 mM CaCl₂) or absence (0.5 mM EGTA) of extracellular Ca²⁺. Data were normalized to intracellular actin levels. The y-axis represents normalized values relative to the values of untreated control cells. (G) Secreted MUC5AC from differentiated control (black circles), KChIP3-GFP (red circles) and KChIP3-MUT (green circles) cells after 30 min incubation at 37°C with vehicle or 10 µM dandrolene (DAND) in the presence of extracellular Ca²⁺. Data were normalized to intracellular actin levels. The y-axis represents normalized values relative to the values of untreated control cells. Abbreviations: EGTA: Buffer with 0.5 mM EGTA, DAND: 10 µM Dandrolene treatment. *p<0.05, **p<0.01, n.s.: not statistically significant.

Figure 2—figure supplement 1. KChIP3 does not control Ca²⁺ levels.

(A) Time course of Ca²⁺ responses (normalized FURA-2AM ratio) (in grey individual cells, in red average trace) obtained in differentiated HT29-18N2 cells in a buffer containing 0.5 mM EGTA to trigger high levels of Ca²⁺ oscillations. After 5 min of recording, cells were treated with vehicle (DMSO) (left) or 10 µM dandrolene (right). At minute 15, cells were stimulated with 100 µM ATP. (B) Percentage of cells oscillating in each condition presented in (A) during 10 min after starting the treatment. Results are average values ± SEM (N > 3). (C) Time course of mean basal intracellular Ca²⁺ levels (normalized FURA-2AM ratio) in KChIP3-KD and control cells. (D) Time course of mean basal intracellular Ca²⁺ levels (normalized FURA-2AM ratio) in KChIP3-GFP and control cells. (E) Time course of mean Ca²⁺ responses (normalized FURA-2AM ratio) obtained in differentiated control (black) and KChIP3-KD (white) cells treated with 100 µM ATP (left panel). Average AUC (Area Under the Curve) from the traces are showed as a bar graph. (F) Time course of mean Ca²⁺ responses (normalized FURA-2AM ratio) obtained in differentiated control (black) and KChIP3-GFP (white) cells treated with 100 µM ATP (left panel). Average AUC (Area Under the Curve) from the traces are showed as a bar graph. (G) Percentage of cells oscillating in KChIP3-KD, KChIP3-GFP and control cells during 10 min in normal buffer (1.2 mM CaCl₂). Abbreviations: Veh: Vehicle (DMSO), Dand: dandrolene. *p<0.05.