Figure 4
Lithium negatively regulates nAChR turnover in CHO-K1/A5 cells. (A) Experimental design used to analyse the effect of lithium on nAChR internalization in CHO-K1/A5. Briefly, cells were incubated overnight with PBS or LiCl at 20 and 50 mM. Then, AlexaFluor555-BTX (BTX1, pseudo-coloured green) was used to label surface nAChRs at 4 °C. After receptors were allowed to internalize for 1 h at 37 °C, AlexaFluor488-BTX (BTX2, pseudo-coloured red) was used to label new nAChRs at the cell surface. To evaluate the effect of lithium on nAChRs density, control- or lithium-treated cells were incubated with AlexaFluor488-BTX for 45 min at 4 °C and then fixed for subsequent fluorescence microscopy visualization. (B) Fluorescence microscopy imaging of CHO-K1/A5 cells labelled with BTX1 and BTX2 revealed the degree of nAChR turnover at the cell surface after 1 h of internalization. (C) Fluorescence microscopy imaging of CHO-K1/A5 cells labelled only with AlexaFluor488-BTX after treatment with LiCl or PBS was performed to determine nAChRs density at the cell surface. Bar = 50 µm. Mean fluorescence intensity for (D) BTX1 or (E) BTX2 at the cell surface after 1 h of nAChR internalization was quantified. Also, (F) BTX1/BTX2 fluorescence intensity ratio at the cell surface was measured and represented in respective graphs as means ± SD along with individual values of two independent experiments. For each group, the membrane-associated fluorescence intensity of 60–80 CHO-K1/A5 cells was analysed. One-way ANOVA and Tukey’s multiple comparisons test were performed for each condition. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.
