CFTR-dependent membrane insertion is linked to stimulation of the CFTR chloride conductance

Abstract

We examined the relation between Cl current (Icl) stimulation and cell membrane capacitance (Cm) when cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in Xenopus oocytes. ICl and Cm increased in parallel when oocytes expressing CFTR were stimulated by forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM). The adenosine 3',5'-cyclic monophosphate (cAMP)-induced increase in surface area detected by Cm was confirmed by morphometry in the same oocytes used for the electrical recordings. These increases in ICl and Cm were reversible and were absent from control oocytes not injected with CFTR cRNA. The time to reach peak ICl lagged slightly behind the peak in Cm. ICl was varied by altering CFTR expression level or agonist dose or by expressing different CFTR mutants. In all cases, there was a close correlation between ICl and Cm, and the kinetics of ICl and Cm stimulation were more rapid the larger the magnitude of the stimulated current. The Cm-ICl relation for wild-type CFTR saturated, consistent with a limited capacity of cells to increase their surface area. These results indicate that stimulation of the CFTR ICl is linked closely to increases in membrane area. This suggests that CFTR is present in the membrane vesicles whose insertion is stimulated by cAMP. The contents of these vesicles may provide a link between activation of CFTR and its cAMP-dependent regulation of other channels.

Cysteine String Protein Interacts with and Modulates the Maturation of the Cystic Fibrosis Transmembrane Conductance Regulator

“…In relation to the initial rationale for these studies, we monitored ⌬C m because Csp is known to function in regulated exocytosis in other systems (21). Control oocytes not injected with CFTR cRNA showed no changes in I m or C m in response to cAMP stimulation, as observed previously (41). Expression of Csp without CFTR also yielded no I m or C m responses to cAMP (data not shown).…”

“…The quality of synthesized cRNA was determined by spectrophotometry and agarose gel electrophoresis. Oocyte preparation and cRNA injections were carried out as described by Takahashi et al (41). Expression proceeded for 2-6 days before current and capacitance recordings, which were performed as described (10,41).…”

“…To initiate such studies, we coexpressed CFTR with either Csp1 or Csp2 in Xenopus oocytes and determined the CFTR-dependent current and capacitance changes evoked during cAMP stimulation. Prior studies of CFTR-expressing oocytes indicate that the cAMP-induced membrane current (⌬I m ) is caused by the chloride flow through the CFTR and that the corresponding increase in membrane capacitance (⌬C m ) monitors changes in cell surface area which are associated with regulated insertion of CFTR into the plasma membrane (10,41). In relation to the initial rationale for these studies, we monitored ⌬C m because Csp is known to function in regulated exocytosis in other systems (21).…”

Cystic Fibrosis F508del Patients Have Apically Localized CFTR in a Reduced Number of Airway Cells

“…It is thus possible that both CFTR and F508del-CFTR lie mostly in the subapical region and that, upon stimulation (eg, by cAMP) CFTR, but not F508del-CFTR, undergoes exocytosis to the apical membrane, as first suggested by Bradbury et al (1992) and also by other authors (Biwersi et al, 1996;Prince et al, 1994;Takahashi et al, 1996;Tousson et al, 1996). The fact that a number of other studies reported apparently contradictory observations (Dho et al, 1993;Dunn et al, 1994;Hug et al, 1997), provides evidence that the issue of CFTR and vesicular trafficking is still an open Double labeling of CFTR, calnexin, p58, and tubulin.…”

μ2 Binding Directs the Cystic Fibrosis Transmembrane Conductance Regulator to the Clathrin-mediated Endocytic Pathway

“…Cell surface biotinylation indicates that CFTR undergoes rapid and efficient internalization in both polarized epithelial and nonpolarized cells (9,10). In addition, several studies indicate that the distribution of CFTR may be regulated by PKA by promoting the translocation of CFTR from an intracellular pool to the plasma membrane and by inhibiting the internalization of CFTR from the plasma membrane (43)(44)(45)(46)(47). These results suggest that the amount of CFTR in the plasma membrane may be regulated by insertion and retrieval mechanisms and that these processes may provide a mechanism to augment PKA activation in regulating CFTR in the plasma membrane.…”