A. Alioua et al., PKG-I-ALPHA PHOSPHORYLATES THE ALPHA-SUBUNIT AND UP-REGULATES RECONSTITUTED GK(CA) CHANNELS FROM TRACHEAL SMOOTH-MUSCLE, American journal of physiology. Lung cellular and molecular physiology, 12(6), 1995, pp. 1057-1063
Modulation of Ca2+-dependent K+ channel (GK(ca)) activities in airway
smooth muscles (ASM) by guanosine 3',5'-cyclic monophosphate (cGMP)-de
pendent protein kinase (PKG) is thought to play a central role in medi
ating the effect of some bronchodilator agents that elevate cytoplas m
ic basal cGMP concentrations. However, no direct evidence supports thi
s hypothesis in ASM. In the present work, we provide evidence that PKG
-I alpha upregulates GK(Ca) channels derived from bovine tracheal smoo
th muscle cells and reconstituted into planar lipid bilayers. In two d
ifferent experimental approaches, PKG increased the open probability a
s well as the mean open time of GK(Ca) channels, without any effect on
unitary current amplitudes and unit conductance. Our results indicate
that the kinetics of GK(Ca) channels are controlled by a phosphorylat
ion step mediated by PKG, and thus might be modulated by intracellular
cGMP. Biochemical assays demonstrated that PKG phosphorylates several
protein bands in the membrane fraction. Two of those proteins co-migr
ate with the same relative molecular mass as the 62- and 30-kDa compon
ents of the purified channel complex, identified as GK(Ca)-alpha and -
beta subunits, respectively. Our results also indicate that PKG phosph
orylates the GK(Ca)-alpha subunit with an apparent stoichiometry of 0.
89, which would be consistent with the presence of a single PKG-sensit
ive phosphorylating site within its amino acid sequence. Furthermore,
these results demonstrate for the first time that PKG directly phospho
rylates GK(Ca) from airway smooth muscle cells and thereby activates t
he channels at negative voltage or at low free Ca2+ concentrations.