CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) is a chloride channel, expressed in the kidney, whose activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study, a possible functional modification of CFTR activity and expression, led by the expression of the hypertensive mutations of adducin (G460W-S586C in humans), an actin capping protein, has been investigated. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild-type or mutated adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing mutated adducin. A higher plasma membrane density of active CFTR channels in the presence of mutated adducin, was confirmed by cell-attached patch-clamp experiments. Western blot and biotinylation experiments demonstrated an increased CFTR expression in the plasma membrane, in the presence of the mutated adducin. FRET (Fluorescence Resonance Energy Transfer) experiments showed a significant but weak interaction between CFTR and adducin, while coimmunoprecipitation experiments failed to reveal a clear interaction between the two proteins. A higher retention of CFTR in the plasma membrane was demonstrated both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that, in HEK cells, the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasma membrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.
MODULAZIONE DEL CFTR DA PARTE DELLA VARIANTE IPERTENSIVA DELLA PROTEINA CITOSCHELETRICA ADDUCINA / F. Sassone ; tutor: G. Meyer; coordinatore: P. Cavallari. - Milano : Università degli studi di Milano. UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Jan 21. ((25. ciclo, Anno Accademico 2012.
|Titolo:||MODULAZIONE DEL CFTR DA PARTE DELLA VARIANTE IPERTENSIVA DELLA PROTEINA CITOSCHELETRICA ADDUCINA|
|Supervisori e coordinatori interni:||CAVALLARI, PAOLO|
|Data di pubblicazione:||21-gen-2013|
|Settore Scientifico Disciplinare:||Settore BIO/09 - Fisiologia|
|Citazione:||MODULAZIONE DEL CFTR DA PARTE DELLA VARIANTE IPERTENSIVA DELLA PROTEINA CITOSCHELETRICA ADDUCINA / F. Sassone ; tutor: G. Meyer; coordinatore: P. Cavallari. - Milano : Università degli studi di Milano. UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Jan 21. ((25. ciclo, Anno Accademico 2012.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/sassone-francesca_phd2013-01-21|
|Appare nelle tipologie:||Tesi di dottorato|