Protein kinase-independent inhibition of muscarinic K+ channels by staurosporine

Abstract

Acetylcholine (ACh) binding to atrial muscarinic receptors activates an inwardly rectifying K+ current (IK[ACh]) via a pertussis toxin-sensitive GTP-binding protein (GK). The muscarinic K+ channel (termed GIRK1) has been cloned, and the nucleotide sequence contains nine consensus sites for protein kinase C (PKC) phosphorylation (16). Dephosphorylation of the muscarinic K+ channel has been implicated in rapid IK[ACh] desensitization in the presence of agonist (13). Staurosporine is a widely used membrane-permeant inhibitor of PKC and other protein kinases (7), including G protein-coupled receptor kinases. We investigated the role of phosphorylation in the regulation of IK[ACh] by examining the effect of a variety of protein kinase inhibitors. Staurosporine produced a rapid and reversible dose-dependent decrease in IK[ACh], activated by either GTP or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Other PKC inhibitors, including calphostin C and K-252b, were without effect on GTP gamma S-activated IK[ACh]. In excised patches of atrial membrane under nonphosphorylating conditions (0 ATP, 1 mM 5'-adenylylimidodiphosphate), staurosporine reversibly reduced muscarinic K+ channel activity without altering single-channel current amplitude. These results suggest that staurosporine inhibits IK[ACh] by a mechanism independent of intracellular protein kinases.

Staurosporine directly blocks Kv1.3 channels expressed in Chinese hamster ovary cells

“…In our experiments, the effects of ST on Kv1.3 current appear to be direct rather than being mediated through PKC or PKA inhibition or other diffusible cytosolic molecules, as indicated by the rapid and direct effect of ST in excised inside-out patches and the inability of PKC and PKA inhibitors to alter Kv1.3 currents in wholecell recordings. Similar direct effects were demonstrated for ST on muscarinic K + channels of atrial myocyte (Lo and Breitwieser 1994) and for calphostin C on L-type Ca 2+ channels of ventricular cells (Hartzell and Rinderknecht 1996). Furthermore, our results show that ST accelerates the rate of Kv1.3 current inactivation.…”

“…Although ST is a relatively specific inhibitor of PKC at low concentrations, it may have actions unrelated to its inhibitory effects of PKC at higher concentrations. For example, in bullfrog atrial myocytes, ST inhibited muscarinic K + channels by a mechanism independent of protein kinase inhibition (Lo and Breitwieser 1994). In the present study, we report the novel pharmacological action of ST that blocks Kv1.3 channels in a PKC-independent manner.…”

Agonist-Selective Mechanisms of μ-Opioid Receptor Desensitization in Human Embryonic Kidney 293 Cells

“…The broad-spectrum protein kinase inhibitor staurosporine (1 M) reduced the opioid-current by 67 Ϯ 12% (n ϭ 3). Such inhibition of GIRK channels by staurosporine has been reported previously, although the mechanism of the effect remains unclear (Lo and Breitwieser, 1994). However, when staurosporine was applied for 10 min before and then during the application of DAMGO (10 M) or morphine (30 M), it did not affect the desensitization induced by DAMGO but it inhibited the desensitization induced by morphine by ϳ60% (Fig.…”

Necessity of protein kinase C activity for maintenance of acetylcholine receptor function at snake twitch fibre endplates

“…Furthermore, because inhibition of kinase activity was not measured directly, we could not rule out the possibility that the decrease in m.e.p.c. recovery by staurosporine was due to a pharmacological action of staurosporine pretreatment unrelated to kinase inhibition (Lo & Breitwieser, 1994). Con-sequently, the present studies were undertaken to examine the effects of several structurally different protein kinase Brkish Joumal of Phannmlogy (1995) 1A [433][434][435][436][437][438][439][440][441] inhibitors on m.e.p.c.…”