Capacitance measurements reveal different pathways for the activation of CFTR

We used the Xenopus laevis oocyte expression system to characterize adenosine 3',5'-cyclic monophosphate (cAMP) activation of the cystic fibrosis transmembrane conductance regulator (CFTR). With conventional two-microelectrode voltage-clamp techniques, we recorded transmembrane conductance G(m) and membrane current (I-m). Using five different sine wave frequencies, we also monitored changes of the plasma membrane surface area by recording continuously membrane capacitance (C-m) under voltage-clamp conditions. Impedance spectra recorded in the frequency range 0.1-500 Hz showed that, at least up to 200 Hz, C-m is independent of the frequency. In control oocytes, cAMP (100 mu M) treatment did not affect G(m) or I-m but evoked a small, slowly occurring increase in C-m, probably mediated by cAMP- stimulated exocytosis. However, in oocytes expressing CFTR, large simultaneous increases of G(m) I-m and C-m occurred after stimulation with cAMP. Oocytes injected with the Delta F508 CFTR mutant behaved like control oocytes and cAMP had no additional effects on G(m) I-m or C-m. In oocytes injected with wild-type CFTR, adenosine 5'-triphosphate (ATP, 100 mu M) did not activate the cAMP-induced augmentation of I-m G(m) or C-m further. On the other hand, cAMP-induced increases in C-m were reduced significantly by the specific blockers of protein kinase A (PKA) KT5720 and N- [2-(methylamino-9-ethyl)]5-isoquinolinesulphonamide hydrochloride (H8), whereas the increases in G(m) and I, were essentially unaffected by these agents. Reducing intracellular Ca2+ by injection of a Ca2+ chelator 1,2-bis (2-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid (BAPTA) prevented PKA-dependent exocytosis while activation of I-m and G(m) of already-inserted CFTR still could be detected. The specific cAMP antagonist adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (Rpc-AMPS) completely suppressed the effects of cAMP on all parameters. These findings are consistent with the concept of different path ways of CFTR activation by cAMP: already-inserted CFTR Cl- channels are activated directly by cAMP, while traffic of CFTR proteins from an intracellular pool to the plasma membrane and functional insertion into the plasma membrane occurs via cAMP- and Ca2+-dependent PKA-mediated exocytosis.