The Large Conductance, Voltage-dependent, and Calcium-sensitive K+ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation in Vivo

Abstract

Native large conductance, voltage-dependent, and Ca 2؉ -sensitive K ؉ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human poreforming ␣-subunit of the Ca 2؉ -sensitive K ؉ channel, Hslo, and the ␣-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by ϳ20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates ␥-33 P into the immunopurified Hslo band of ϳ125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the ␣-subunit of large conductance Ca 2؉ -sensitive K ؉ channels is substrate for G-I␣ kinase in vivo and support direct phosphorylation as a mechanism for PKG-I␣-induced activation of maxi-K channels.

Leucine Zipper Domain Targets cAMP-dependent Protein Kinase to Mammalian BK Channels

“…In these assays we stimulated PKA activity intimately associated with the channel by applying cAMP to the intracellular face of isolated inside-out patches. As previously reported (17) the effects of cAMP in this system are dependent upon the presence of Mg-ATP, and the actions of cAMP are completely abolished by the PKA inhibitor peptide PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] . Although STREX channels are inhibited whereas ZERO channels are activated by PKA closely associated with the channel (17), the short LZ1-competing peptides effectively blocked PKAdependent regulation of either splice variant.…”

Persistent Changes in Spontaneous Firing of Purkinje Neurons Triggered by the Nitric Oxide Signaling Cascade

“…In smooth muscle tissue, BK channels are facilitated by NO-cGMP (Archer et al, 1994). PKG has been reported to phosphorylate BK channels directly, and this modification shifts the voltage-activation curve to more hyperpolarized potentials (Alioua et al, 1998). Furthermore, it has been shown in pituitary nerve terminals that PKG activation increases channel open probability, and this modification seems to increase excitability, leading to more action potentials in response to current injection (Klyachko et al, 2001).…”

Hypercontractility and impaired sildenafil relaxations in the BKCachannel deletion model of erectile dysfunction

“…The importance of this kinase in erectile tissue is supported by the observation that precontracted corpus cavernosum smooth muscle (CCSM) strips from mice lacking PKGI did not relax to nerve stimulation (14). It is known that PKGI activates large-conductance Ca 2ϩ -activated potassium (BK Ca ) channels (1,32), which hyperpolarize smooth muscle cell membranes, causing muscle relaxation. Relaxation of arterial and CCSM is necessary to increase blood flow into the corpora cavernosa that leads to penile tumescence.…”