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All authors read and approved the final manuscript

All authors read and approved the final manuscript. Acknowledgements We thank Professors C. at 1?mM glucose (1G) or 16.7?mM glucose (16.7G) in the absence or presence of FSK, GLP-1, CFTRinh-172 (CFTRinh) and GlyH-101 (GlyH) as indicated (n?=?12 to 21, N?=?4 to 9). (E) Insulin secretion from mouse islets at 1?mM glucose (1G) in the absence or presence of FSK and inhibitors as indicated (n?=?9C12, N?=?4). (F) Insulin secretion from mouse (left) and human (right) islets in the absence of FSK to demonstrate the lack of effect of the inhibitors (mouse: n?=?10, N?=?5; human n?=?12, N?=?3). (G) Localization of CFTR (yellow) and insulin (red) in fixed single islet cells (left) from human (top) and mouse (bottom), detected using confocal immunocytochemistry. Scale bar 5?m. Images are representative of 37 beta-cells from three human donors and 23 beta-cells from three mice. Ratio of the fraction of CFTR (right) in the plasma membrane region (P1) as compared to the cytosolic region (P2) for human (top) and mouse (bottom) beta-cells. Data are presented as mean??SEM. ***<0.001 16.7?G 1?G, ??<0.01 FSK or GLP-1 respective G alone, ???<0.001 FSK respective G alone and ?<0.05 CFTRinh or GlyH 16.7?G and FSK alone, ???<0.001 GlyH 16.7G and FSK alone. The presence of active CFTR channels in pancreatic beta-cells was investigated on single cells using the patch-clamp technique in the standard whole-cell configuration. The pipette solution contained sodium and calcium ions in order to determine the cell-type by sodium channel inactivation properties [32]. A voltage-ramp protocol from ?100?mV to +100?mV was applied before and every fourth minute after the addition of FSK (10?M) until steady state was achieved (Figure?2). In the absence of FSK the current flow was minimal, whereas the increase in intracellular cAMP induced by FSK activated a non-linear outward rectifying current. In human and mouse beta-cells, the cAMP-activated current was significantly inhibited by the CFTR-inhibitors (Figure?2A-D). The current inhibited by CFTR-inhibitors (CFTR-dependent) constitute 47??15% (n?=?7) and 57??7% (n?=?10) of the FSK-activated current at negative potentials, in human and mouse beta-cells, respectively. Open in a separate window Figure 2 cAMP-activated chloride currents in human and mouse beta-cells. (A) Currents measured in a single human beta-cell after stimulation with voltage ramps in the absence (Ctrl, light gray) and presence of forskolin (FSK; gray), in the simultaneous presence of FSK and GlyH-101 (FSK and GlyH; black) and after wash-out of GlyH-101 to recover the FSK-activated current (WO: FSK; dark gray). Current ramps were applied before and every fourth minute after the application of FSK until a steady state was achieved. (B) Bar graph of the membrane conductance at negative voltages (left; n?=?7 to 17, N?=?3) and graph of calculated FSK-activated and CFTR-dependent current (right; Mean of n?=?7 cells) from data in A. (C) Same as in A, but experiments where performed on mouse beta-cell. GlyH-101 (GlyH: black trace) and CFTRinh-172 (CFTRinh, black) was added to the left and right, as indicated. (D) As in B, but membrane conductance (left) was calculated from data in C (n?=?10 to 17, N?=?8). The mean result was combined for both CFTR-inhibitors PDGFB (Inh). The calculated FSK-activated and CFTR-dependent current to the right is a mean from 10 cells. (E) As in A, but the effect of 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) was investigated (n?=?6, N?=?2). Calculated FSK-activated, DIDS-sensitive and CFTR-currents shown to the right are mean of n?=?5 cells. (F) Same as in E, but the membrane conductance (left) was calculated from FAA1 agonist-1 measurements in mouse beta-cells (n?=?9, N?=?6) and the calculated current to the right is the mean from n?=?8 cells. Data are presented as mean??SEM. *<0.05, ***<0.005, ?<0.05, ???<0.005, ?<0.01 and ??<0.01. In addition to the ion FAA1 agonist-1 channel function, CFTR has been attributed a role as regulator of other ion channels and proteins, such FAA1 agonist-1 as other chloride channels [2,33]. To investigate the possibility that CFTR FAA1 agonist-1 regulates the function of other chloride channels we used the non-specific chloride channel blocker DIDS that blocks a wide variety of chloride channels, while CFTR.