AJP - Cell Physiology, Vol 262, Issue 5 C1304-C1312, Copyright © 1992 by American Physiological Society
cGMP-dependent protein kinase regulation of a chloride channel in T84 cells
M. Lin, A. C. Nairn and S. E. Guggino
Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Chloride channels at the apical membrane of intestinal epithelial cells are
involved in the excessive fluid secretion in diarrhea and diminished
secretion in cystic fibrosis (CF). Diarrhea induced by heat-stable toxin
from Escherichia coli is associated with elevated guanosine 3',5'-cyclic
monophosphate (cGMP) in intestinal epithelial cells, but it is unknown
whether chloride secretion is regulated by cGMP directly or via
cGMP-dependent protein kinase (PKG). Single-channel recordings (inside-out
excised patches) from the apical membrane of T84 cells reveal a 10-pS
chloride channel with a linear current-voltage relationship, which is
opened when an endogenous membrane-bound PKG is activated with ATP (1 mM)
and cGMP (100 microM). Soluble PKG (200 nM) isolated from bovine lung,
added to the intracellular face of patches, also opens this channel. No
activation occurs with Ringer solution alone or only ATP or cGMP. Addition
of nonhydrolyzable forms of ATP (AMP-PNP, 1 mM) or a combination of ATP,
cGMP, plus H-8 (5 microM), an inhibitor of PKG, also does not stimulate the
channel. The catalytic subunit of adenosine 3',5'-cyclic
mono-phosphate-dependent protein kinase (PKA, 200 nM, with 1 mM ATP)
activates a channel with similar characteristics. The 10 pS channel has a
PNa/PCl ratio of 0.06, an anion selectivity of Br- (1.2) greater than Cl-
(1.0) greater than I- (0.8) greater than F- (0.4), and a low affinity for
the chloride channel blockers, 4,4-dinitrostilbene-2,2-disulfonic acid and
5-nitro-2-(3-phenylpropylamino)benzoic acid.(ABSTRACT TRUNCATED AT 250
WORDS)
