Chloride channels play key roles in pancreatic beta-cells [17, 52–54], but the exact identity of all chloride channels involved remains to be established. A previous study indicated that endocrine cells in the rat pancreas express the mRNA encoding CFTR . Here we provide novel data adding CFTR to the list of chloride channels having important functions in human and mouse beta-cells [17, 52–54], and suggest that CFTR acts upstream of ANO1 [2, 33, 41] to control insulin secretion.
In a previous study, Kinard and Satin  measured an ATP- and cAMP-dependent chloride current in insulin secreting cells that could be activated under hypotonic conditions. The current was termed ICl,islet and had similar properties in terms of size and reversal potential as the chloride current obtained here in the presence of glucose and FSK/GLP-1 (Figure 2). It was suggested  that the ICl,islet contributes to cAMP-dependent depolarization at negative membrane potentials although the exact identity of the channel was not described. It has been suggested that the channel is a volume-regulated anion channel (VRAC; ), due to similarities with ICl,islet in terms of activation and electrophysiological properties. Our focus was CFTR and we did not investigate the presence of a current sensitive to cell swelling. However, our data do not rule out the presence of VRAC. Whereas VRAC is suggested to enhance electrical activity, we hypothesize that the main function of CFTR/ANO1 is in the control of exocytosis. We confirm that activation of the ATP- and cAMP-dependent chloride current contributes to a small depolarization at potentials below the equilibrium potential for chloride, when the flux of chloride ions is outward from the cell comparable to the depolarization obtained by cAMP on electrical activity measured on whole islets [56, 57].
The measured current conductance in the presence of FSK varies among different batches of cells investigated and amounts between approximately 80 and 120 pS/pF and approximately 20 and 40 pS/pF in mouse beta-cells (Figures 2B, F and 3F) and human beta-cells (Figure 2B, E), respectively. Biological variation is common in studies on primary tissue and human primary tissue in particular. However, more importantly, the estimated CFTR conductance after blockade of other chloride-currents using DIDS or by blocking ANO1 becomes the same (13 pS/pF and 12 pS/pF, respectively), strongly supporting the presence of a CFTR conductance. Although small, the impact on insulin secretion is large, suggesting that CFTR has a function upstream of many other cAMP-activated processes involved in insulin secretion [19, 27, 49, 56, 58].
We provide evidence suggesting that the main consequence of the cAMP-activated chloride current is unrelated to membrane depolarization (Figure 4A). More specifically our studies point to a role of CFTR and ANO1 in cAMP augmented calcium-dependent exocytosis. Indeed, both FSK- and GLP-1-enhanced, glucose-stimulated insulin secretion is reduced by CFTR-inhibitors. We observed that the inhibitors are more potent on GLP-1 than on FSK-stimulated secretion. This we mainly attribute to the fact that FSK increases cAMP to a higher level than GLP-1 in islets (see, for example, ). The participation of CFTR in the exocytotic process is supported by the capacitance measurements showing that exocytosis is blocked by CFTR-inhibitors in both human and mouse beta-cells (Figure 4). Exocytosis is a calcium-dependent process and the reduced exocytotic response observed in the presence of the CFTR antagonists might be explained by a reduced calcium current , but this was proven not to be the case (Figure 4F, G). Based upon our observations from insulin secretion and electrophysiological ramp-protocol measurements we instead hypothesize that CFTR through ANO1 act directly on exocytosis. Indeed, we found CFTR to co-localize with the SNARE-protein syntaxin 1A (Figure 5E) as demonstrated in other tissues [50, 51]. This suggests that CFTR, as syntaxin 1A, is part of the exocytotic machinery.
From our novel results we suggest that CFTR plays a key role in priming of the insulin granules; this is evident from the capacitance measurements and the electron micrographic data. The increase in membrane capacitance evoked by a train of membrane depolarizations initiates exocytosis of release-ready (primed) granules by the first two depolarizations, whereas the latter depolarizations enable granules within a larger reserve pool to be released . Our data demonstrate a pronounced reduction in exocytosis evoked during the two first depolarizations (Figure 4B-E). Hence, CFTR can be suggested to have its main function in the priming of insulin granules. Moreover, the ultrastructural analysis revealed that the number of granules in close vicinity to the plasma membrane (<300 nm) was reduced after CFTR inhibition (Figure 5D). The exact molecular mechanism by which CFTR contributes to granular priming is hitherto unknown. Interestingly, DIDS has earlier been demonstrated to reduce insulin exocytosis, an effect that has been coupled with intragranular ClC3 chloride channels and priming of the insulin granules . A role for ClC3 in insulin granular priming and exocytosis has also been proven by knock-out animals . Here we demonstrate that CFTR inhibition reduces exocytosis to the same extent as has earlier been demonstrated for DIDS  and removal of ClC3 . As DIDS is a chloride channel blocker inhibiting the current through most chloride channels except for CFTR, the ANO1 current is likely reduced by this treatment (compare Figures 2F and 3F). Our insulin secretion measurements and chloride current measurements suggest that CFTR acts on ANO1. The mechanisms by which ANO1 is regulated by CFTR is yet to be investigated, but the small influx of chloride through CFTR seems to have some function since the ANO1 current was inhibited by the CFTR-specific open-channel blocker Gly-H 101 (Figure 3F). The fact that ANO1 is calcium activated  is consistent with a role of this channel in exocytosis. It can be argued that the increase in cAMP will enhance the voltage-dependent calcium-influx  and thereby increase the ANO1 current and insulin secretion. This process can most likely occur in parallel with the cAMP-dependent regulation of ANO1 via CFTR as demonstrated here. Our data, however, demonstrate a role for CFTR in regulating insulin secretion through a direct effect on exocytosis.