Regulation of CFTR and its contribution to other epithelial Cl- channels
Year: 2011
Type: dissertation
Abstract: Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a cAMP dependent Cl- channel that is expressed mainly on the apical membrane of epithelial cells. CFTR plays an essential role in electrolyte and water homeostasis. Defects in CFTR cause CF disease, which is the most common lethal genetic disorder among Caucasians. Besides functioning as a Cl- channel, CFTR also acts as a regulator of other channels and transporters. Although CFTR has been known for more than 20 years, many questions regarding CFTR regulations and functions remain to be answered. In the present thesis, we performed various experiments in an attempt to understand CFTR in several aspects, including CFTR-regulation by AMPK in vitro and in vivo, the role of the S573C CFTR mutation for the risk to develop pancreatitis, and a novel role of CFTR in proton sensing and regulating CaCC. Regulation of CFTR by AMPK in vivo The present study has clarified the role of AMPK for epithelial Cl- transport in vivo using AMPKα1-/- mice. The data demonstrate a significant role of AMPK in regulating CFTR. In real short circuit Ussing chamber experiment, an increase in CFTR Cl- current is observed in the colon of AMPKα1-/- mice when exposed to cAMP. Unlike wild type littermates, the elevated cAMP-dependent Cl- current in the AMPKα1-/- colon is insensitive to the AMPK activator phenformin. Furthermore, rectal potential difference (RPD) measurements indicate that AMPKα1-/- mice have an increased RPD both non-stimulated and after exposure to cAMP. These data suggested enhanced activity of CFTR in the large intestine of AMPKα1-/- mice. In correlation to previous in vitro observations, data from the present study demonstrate that AMPK inhibits CFTR in vivo. Mechanistic insight into control of CFTR by AMPK To identify the AMPK phosphorylation sites in CFTR and to find the mechanisms underlying AMPK regulation of CFTR, Xenopus oocytes were used to overexpress different CFTR mutations, and the whole cell currents of the overexpressed oocytes were measured using the double electrode voltage clamp (DEVC) technique. In contrast to previous observations, this study demonstrates that S737 and S768 are phosphorylated by AMPK rather than PKA. In Xenopus oocytes, single or multiple mutations of S737 and S768 to alanine increase CFTR whole cell currents around 4-fold. Moreover, in non-activated cells, S737A and S768A increase the basal CFTR Cl- conductance around 5-fold. The elevated conductance is insensitive to the AMPK inhibitor phenformin and the AMPK activator compound C. Taken together, we demonstrate that AMPK inhibits PKA activation of CFTR. Moreover, AMPK indeed constitutively phosphorylates CFTR and keeps the channel shut at basal levels. We associated this finding to a spatiotemporal regulation of CFTR by cAMP. Data from the present study suggest a constitutive inhibition of CFTR by a tonic baseline AMPK activity. Because AMPK can also be activated without any detectable changes in the global AMP/ATP ratio, it is likely that AMPK regulation of CFTR is also controlled locally. The scenario can perhaps be described as AMP being generated by a Shank2/PDE complex bound to CFTR’s PDZ domain. PDE degrades cAMP to AMP, increasing the AMP/ATP ratio close to CFTR, and thus activating AMPK. The activated AMPK therefore inhibits CFTR and keeps the channel close under non-stimulated conditions. Metformin treatment of diabetes mellitus increases the risk of pancreatitis in patients bearing the CFTR-mutation S573C Metformin is an AMPK activator and is a drug used for the treatment of diabetes type II. Lactic acidosis is a common secondary complication of metformin therapy, especially in patients with renal dysfunction, alcohol abuse, or liver disease. Patients with renal failure have been reported to develop the pancreatitis after metformin therapy. We examined the effects of metformin treatment on a pancreatitis-related S573C-CFTR mutation. The results demonstrate a slight but significant reduction of S573C-CFTR whole cell currents that are further inhibited by metformin. On the other hand, metformin treatment of wtCFTR does not affect whole cell current induced by cAMP. These data suggest that the S573C mutation increases the sensitivity towards AMPK, However in vitro phosphorylation experiments reveal the same pattern of AMPK phosphorylation in wt and S573A-CFTR. Because lactic acidosis has been found during metformin therapy, and because CFTR is essential to control the pH balance in the pancreas, we challenged wt and S573C CFTR expressing oocytes by exposing them to acidic pH. The results demonstrate that intracellular acidificaiton reduces activation of wtCFTR, but almost completely abolishes Cl- currents produced by S573C-CFTR. Taken together these data imply that patients carrying the S573C mutation have only a slight defect in CFTR Cl- currents. However, under metformin treatment, the Cl- transport in patients carrying the S573C mutation is largely reduced. Thus S573C-carrier have a higher risk in developing a pancreatitis after metformin therapy. CFTR induces acid sensing and H+ activated Cl- transport In the last section of the present thesis we identified a novel role of CFTR for detection of the extracellular proton concentration and regulation of the Ca2+ activated Cl- current. In wtCFTR expressing Xenopus oocytes, we observed a Ca2+ activated Cl- current after exposure of the oocytes to extracellular acidic pH. This current was strongly outwardly rectifying, sensitive to DIDS and NPPB, and required intracellular Ca2+ for the activation. We hypothesize that CFTR translocate H+-receptors to the plasma membrane and extracellular protons binding to these receptors induces an increase in intracellular [Ca2+]. We speculate that CFTR-dependent H+-sensing may be essential for bone metabolism, since CFTR is expressed in bone cells and because CF-patients suffer from osteoporosis.
DOI: 10.5283/epub.20683
Author: Kongsuphol, Patthara
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W172580192