ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC)

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca(2+)-activated Cl(−) channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca(2+) and voltage-dependent Cl(−) currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16A(inh)-A01, CaCC(inh)-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00424-014-1598-8) contains supplementary material, which is available to authorized users

Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca²⁺

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6’s physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich–Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca(2+) is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium

Hans H. Ussing - scientific work: contemporary significance and perspectives

AbstractAs a zoologist, Hans H. Ussing began his scientific career by studying the marine plankton fauna in East Greenland. This brought him in contact with August Krogh at the time George de Hevesy, Niels Bohr and Krogh planned the application of artificial radioactive isotopes for studying the dynamic state of the living organism. Following his studies of protein turnover of body tissues with deuterium-labeled amino acids, Ussing initiated a new era of studies of transport across epithelial membranes. Theoretical difficulties in the interpretation of tracer fluxes resulted in novel concepts such as exchange diffusion, unidirectional fluxes, flux-ratio equation, and solvent drag. Combining methods of biophysics with radioactive isotope technology, Ussing introduced and defined the phrases ‘short-circuit current’, ‘active transport pathway’ and ‘shunt pathway’, and with frog skin as experimental model, he unambiguously proved active transport of sodium ions. Conceived in his electric circuit analogue of frog skin, Ussing associated transepithelial ion fluxes with the hitherto puzzling ‘bioelectric potentials’. The two-membrane hypothesis of frog skin initiated the study of epithelial transport at the cellular level and raised new questions about cellular mechanisms of actions of hormones and drugs. His theoretical treatment of osmotic water fluxes versus fluxes of deuterium labeled water resulted in the discovery of epithelial water channels. His discovery of paracellular transport in frog skin bridged studies of high and low resistance epithelia and generalized the description of epithelial transport. He devoted the last decade of his scientific life to solute-coupled water transport. He introduced the sodium recirculation theory of isotonic transport, and in an experimental study, he obtained the evidence for recirculation of sodium ions in toad small intestine. In penetrating analyses of essential aspects of epithelial membrane transport, Ussing provided insights of general applicability and powerful analytical methods for the study of intestine, kidney, respiratory epithelia, and exocrine glands—of equal importance to biology and medicine