Df508-cftr channels: kinetics, activation by forskolin and
potentiation by xanthines.
Haws, C. M., I. Nepomuceno, M. E. Krouse, H. Wakelee, T. Law, Y. Xia,
H. Nguyen, and J. J. Wine.
Cystic Fibrosis Research Laboratory, Stanford University, Stanford,
CA 94305-2130, Johns Hopkins School of Medicine, Baltimore, MD 21205,
Megabios Corp., 863A Mitten Road, Burlingame, CA 94010
APStracts 2:0392C, 1995.
Trafficking, activation and kinetics of DF508-CFTR and CFTR were
compared in stably transduced C127 mouse mammary epithelial cells.
Western blots detected a small amount of fully glycosylated DF508
-CFTR. Efflux of 125I was stimulated by forskolin with the same EC50
(0.5 [mu]M) for CFTR and DF508-CFTR cells, but the maximum response
was reduced >5 fold and its latency increased 3-fold in DF508
-CFTR cells. In DF508-CFTR cells, IBMX (EC50 = 1.45 mM) and CPX (EC50
= 58 [mu]M) increased the peak forskolin-stimulated efflux rate about
2.5 fold and decreased the time-to-peak. A 7-fold increase in
intracellular cAMP levels accompanied potentiation of forskolin
-induced 125I efflux by IBMX, but not by CPX. Elevation of
intracellular cAMP increased linear, voltage-independent whole-cell
currents 30-fold in CFTR and 4-fold DF508-CFTR cells; the response
rate in DF508-CFTR cells was much slower. Single channel currents
were detected in 57/68 cell-attached patches from forskolin pre
-stimulated CFTR cells vs. 6/35 patches in DF508-CFTR cells. Mean
number of active channels/patch was 4.1 for CFTR (Po = 0.34) and 0.2
for DF508-CFTR (Po = 0.11). The lower open probability of DF508-CFTR
resulted from a 3-fold longer mean interburst interval. We estimate
that forskolin-stimulated chloride conductance of DF508-CFTR C127
cells is <5% of CFTR cells. CPX is 25 fold more potent than IBMX
in potentiating DF508-CFTR, and may operate by a mechanism other than
elevation of cAMP.
Received 17 July 1995; accepted in final form 20 October 1995.
APS Manuscript Number C430-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 November 95