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. 2014 Jul 15;5:4420. doi: 10.1038/ncomms5420

Figure 2. Involvement of CFTR-medicated Cl− efflux in maintaining resting membrane potential of β-cells.

Figure 2

(a) CFTR inhibitors, CFTRinh-172 (10 μM) and glyH-101 (10 μM), induced membrane hyperpolarization in RINm5F cells assessed by voltage-sensitive fluorometric measurement with Dibac on RINm5F cells. Number of measurements is shown in each column. (b) Intracellular chloride measurement with MQAE chloride-sensitive fluorescent dye in RINm5F cells (left panel: calibration curve, right panel: time course of changes in [Cl−]i). Application of CFTRinh-172 (10 μM) led to elevation of [Cl−]i by 25.9±1.3 mM (n=36). (c) Membrane potential measurement by patch-clamp in RINm5F cells. CFTRinh-172 (10 μM) led to hyperpolarization of the membrane. **P<0.01, t-test. The experiment was repeated five times. (d) Membrane potential of β-cells measured by patch-clamp. Freshly isolated β-cells from DF508 mutant mice (n=4) had more negative resting membrane potential than that from wild-type mice (n=5). *P<0.05, t-test. (e) KATP channel inhibitor glibenclamide (GLIB, 1 μM) depolarized the membrane (left), whereas in the presence of glyH-101 (10 μM), GLIB failed to induce depolarization (right) in RINm5F cells. Number of measurements is shown in each column of the summary chart. ***P<0.001, one-way analysis of variance (ANOVA). (f) Glucose (10 mM) induced depolarization by about 20 mV in RINm5F cells. Pretreatment with CFTRinh-172 (10 μM) and glyH-101 (10 μM) inhibited the glucose-induced depolarization. Number of measurements is shown in each column. ***P<0.001, one-way ANOVA. (g,h) Recording of action potentials evoked by current injection in RINm5F cells; (g) Knockdown of CFTR (CFTRkd) decreased the action potential evoked by 0.05 and 0.3 nA currents, as compared with LacZ control, with number of experiments shown in data bars. *P<0.05, t-test. Western blots show the protein level of CFTR after knockdown. Uncropped immunblot is shown in Supplementary Fig. 8. (h) CFTRinh-172 (10 μM) completely abolished the action potential evoked by 0.05 nA current stimulus and partially abolished the action potential evoked by 0.3 nA current stimulus with number of experiments shown in corresponding data bars. *P<0.05, **P<0.01, t-test. [Cl−]o=142 mM (a–h). [Cl−]i=98 (a–f) and 150 (g,h) mM. ECl= −9.8 (a–f) and 1.4 (g and h). Data are shown as mean±s.e.m.