Mechanism of activation by cyclic gmp-dependent protein kinase of
large, ca2+-activated k+ channels in human glomerular mesangial cells
.
Stockand, James D., and Steven C. Sansom.
Division of Renal Diseases and Hypertension and Department of
Integrative Biology, University of Texas Medical School at
Houston
APStracts 3:0188C, 1996.
The patch clamp method was employed to establish the mechanism of
regulation by dibutyryl-cGMP (db-cGMP) and protein kinase of large,
calcium-activated K channels [BK(Ca)] in human mesangial cells (MC).
In cell attached patches (CA), db-cGMP significantly increased open
probability (Po) of BK(Ca), however Po was unaffected by addition of
db-cAMP or db-cGMP plus staurosporine, a general kinase inhibitor. In
excised, inside-out (I/O) patches, BK(Ca) was activated upon addition
of Mg-ATP plus db-cGMP; however, cAMP plus Mg-ATP, or separate
additions of db-cGMP and Mg-ATP failed to activate BK(Ca). Thus, the
endogenous kinase activity is specific for db-cGMP and associated in
I/O patches with BK(Ca). Activation by db-cGMP plus Mg-ATP of BK(Ca)
in I/O patches was blocked by KT5823, a specific inhibitor of cGMP
-dependent protein kinase (PKG) but not by KT5720, a specific
inhibitor of cAMP-dependent protein kinase (PKA). These results
demonstrated that the endogenous protein kinase was a member of the
cGMP-dependent family. Protein kinase G plus db-cGMP and Mg-ATP
activated BK(Ca) from 0.07 to 0.39; in contrast, BK(Ca) was not
affected by Mg-ATP plus either the catalytic subunit of PKA or PKC
and OAG. PKG activated BK(Ca) by decreasing the long, closed kinetic
time constant from 192 to 22 ms. The half-activation potential
(V1/2), defined as the potential at which the open probability is 0.5
with 1 [mu]M Ca, was -34 and 42 mV for the activated and unactivated
channels, respectively; however, the gating charge (zg), a measure of
voltage sensitivity, was not significantly affected by PKG.
Similarly, whereas the Ca1/2, defined as the [Ca]i required to
activate BK(Ca) to Po = 0.5 at 40 mV, decreased from 1.74 to 0.1 _M
upon addition of PKG, the Hill coefficient, which describes Ca2+
sensitivity, was not affected. A change in V1/2 and Ca1/2, but not zg
and the Hill coefficient, is consistent with a PKG-dependent change
in activation threshold, which is independent of voltage and calcium
sensitivities. PKG activation of BK(Ca) was heterogenous with two
populations: a majority (approximately 67%) of BK(Ca) were activated
in CA by db-cGMP or in I/O by either db-cGMP or PKG plus Mg-ATP;
however, a minority (approximately 33%) of BK(Ca) could not be
activated by these compounds. Addition of the protein phosphatase
(PP) inhibitor, okadaic acid (OA), did not influence PKG-dependent
activation of BK(Ca). It is concluded that an endogenous PKG
activates BK(Ca) by decreasing the Ca and voltage activation
thresholds.
Received 26 December 1995; accepted in final form 9 May 1996.
APS Manuscript Number C759-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 28 June 96