Hypertension-Linked Mutation of α-Adducin Increases CFTR Surface Expression and Activity in HEK and Cultured Rat Distal Convoluted Tubule Cells

Abstract

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-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 the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT 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 plasmamembrane. 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.

G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2

“…2B), as confirmed by calculation of the half time of recovery (t ½ ), after fitting values to one phase decay curves37: t ½ values of LANCL2-PAGFP and of LANCL2-G2A-PAGFP were 50 ± 30 s and 3.4 ± 0.75s, respectively (graph in Fig. 2B; =34 cells from four different transfection experiments, p < 0.001).…”

“…Images acquired at the same time points after photoactivation clearly indicated that the fluorescence decay of LANCL2-PAGFP was much slower than that of LANCL2-G2A-PAGFP ( Fig. 2B ), as confirmed by calculation of the half time of recovery (t ½ ), after fitting values to one phase decay curves 37 : t ½ values of LANCL2-PAGFP and of LANCL2-G2A-PAGFP were 50 ± 30 s and 3.4 ± 0.75s, respectively (graph in Fig. 2B ; =34 cells from four different transfection experiments, p < 0.001).…”

“…At each time point the mean fluorescence value (F t ) in the bleached ROI was analysed as described 37 55 : each ROI F t was normalized for the F t of a ROI not subjected to photobleaching, to correct for photobleaching of EGFP due to the imaging procedure. Photobleaching-corrected F t values were subtracted of first post-photobleaching values and normalized for the mean fluorescence value measured before photobleaching.…”

HEK-293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl−transport

“…On the other hand, Mondini et al. () reported that CFTR inh 172 could completely block the whole cell current attributable to transiently transfected CFTR in these HEK‐293 cells. The variability in efficacy of CFTR inh 172 could either be due to differences in the source of the inhibitor, the relative insolubility of the compound in aqueous solution, or in the structure function relationship of the EGFP‐CFTR protein.…”

“…However, Mondini et al. () used HEK‐293 cells to examine the effect of a hypertension‐linked mutation of α ‐adducin on CFTR surface expression and channel activity. While in these studies, the HEK‐293 cells were stably transfected with wild type or mutant α ‐adducin, they were transiently transfected with a bicistronic vector, pIRES2‐CFTR‐EGFP, which expressed separate EGFP and CFTR proteins.…”