cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in stage V/VI Xenopus oocytes by injection of cRNA transcribed in vitro from a pBluescript vector containing 6.2-kb wild-type cDNA. This clone was also used for the preparation of antisense RNA. Double-electrode voltage clamp was employed to measure transmembrane currents. In sense RNA-injected oocytes, cAMP depolarized the membrane potential (Vm) from -52 to -31 mV and increased membrane conductance (Gm) 10-fold. However, cAMP had no effect on Vm or Gm in uninjected oocytes or in oocytes injected with antisense RNA. The endogenous Ca-activated Cl currents of control oocytes were abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50 microM) or bath Cl replacement. In contrast, the cAMP-stimulated currents of CFTR-expressing oocytes were DIDS insensitive and were inhibited only approximately 50% when bath Cl was replaced by gluconate or glutamate. In addition, the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; 50 microM) and diphenylamine-2-carboxylic acid (DPC; 3 mM) reduced the cAMP-evoked currents by only approximately 10%. The stimulated currents of CFTR-expressing oocytes were reduced approximately 30% by 10 mM Ba, suggesting that the Cl-independent current component is due to an increase in K conductance. Our results indicate that expression of CFTR in Xenopus oocytes produces a cAMP-activated Cl current. The Cl-independent current may represent a regulatory action of CFTR on K conductance pathways or a secondary response of the oocyte membrane to the high Cl conductance induced by CFTR expression.

Mechanisms of chloride secretion induced by thermostable direct haemolysin of Vibrio parahaemolyticus in human colonic tissue and a human intestinal epithelial cell line

“…5a). Glybenclamide (300 ìM), an inhibitor of cAMPdependent Cl À channels [28,29], 100 ìM NPPB, an inhibitor of Cl À channels [26,28] and gadolinium 3 , an inhibitor of the stretch-activated channel [30,31], had no effect on TDH-induced increases in Isc (Fig. 5c).…”

“…Fig. 5a shows that the Isc increase in Caco-2 monolayers stimulated by TDH was inhibited by DIDS (100 ìM), an inhibitor of Ca 2 -activated Cl À channels [26,27]. When Caco-2 cell samples were pretreated with DIDS, TDH had no effect on Isc (Fig.…”

“…The Isc did not change when PBS was added. The high Isc induced by TDH decreased when 100 ìM DIDS, an inhibitor of Ca 2 -activated Cl À channels [26,27], was added. These results suggest that TDH increases Cl À secretion by human colonic tissues.…”

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

“…This is not observed in epithelial cells from CF patients in response to cAMP or cAMP-stimulating agents. ⌬ F508-CFTR forms PKA-activated chloride channels in lipid bilayers (1), in oocytes (2,3), and in the ER membrane (4), but is prevented from reaching the epithelial cell surface (5). To study ⌬ F508-CFTR trafficking, we chose the IB3-1 cell line, an immortalized CF bronchial epithelial cell line with genotype ⌬ F508/ W1282X (17).…”

“…This mutation results in a protein capable of conducting chloride (1)(2)(3)(4), but absent from the plasma membrane because of aberrant intracellular processing (5). Under usual conditions at 37 Њ C, ⌬ F508-CFTR is retained in the endoplasmic reticulum (ER), perhaps by prolonged association with the ER chaperones calnexin (6) and hsp70 (7), and is then targeted for degradation by the ubiquitin/proteasome pathway (8,9).…”