NDLI: Capacitance measurements reveal different pathways for the activation of CFTR

Content Provider	Springer Nature Link
Author	Weber, W. M. Cuppens, H. Cassiman, J. J. Clauss, W. Van Driessche, W.
Abstract	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 µM) 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 ΔF508 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 µM) 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 m were essentially unaffected by these agents. Reducing intracellular Ca2+ by injection of a Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,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 (RpcAMPS) completely suppressed the effects of cAMP on all parameters. These findings are consistent with the concept of different pathways 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.
Starting Page	561
Ending Page	569
Page Count	9
File Format	PDF
ISSN	00316768
Journal	Pflügers Archiv
Volume Number	438
Issue Number	4
e-ISSN	14322013
Language	English
Publisher	Springer-Verlag
Publisher Date	1999-07-20
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Content Type	Text
Resource Type	Article
Subject	Physiology Physiology (medical) Clinical Biochemistry

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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