Inhibition by wortmannin of M‐current in bullfrog sympathetic neurones

Abstract

1 The actions of wortmannin, an inhibitor of myosin light chain kinase (MLCK), on M-type potassium current of dissociated bullfrog sympathetic neurones have been examined. 2 The amplitude of M-current was measured by whole cell recordings from cells pretreated with wortmannin (0.01-10gM) or the wortmannin vehicle, dimethylsulphoxide (0.0001-0.1 vol%), for 30 min. Internal (recording pipette) solutions having three different pCa values (6, 7 and 8) were used for the measurements. 3 Irrespective of the pCa, M-current was not detectable when the cells were pretreated with 10 AM wortmannin. Wortmannin, 3 tLM, produced 85-95% inhibition of the M-current. Pretreatment with 10-30 nM wortmannin was without effect on M-current. 4 The M-current inhibition by wortmannin at concentrations of 0.1-1 gM depended on the pCa of the internal solution. Inhibition occurred only when the calcium-rich (pCa = 6) internal solution was used. 5 Pre-treatment of the cells with wortmannin (10 IM) did not affect rapidly-inactivating A-type or delayed rectifier-type potassium currents nor did it alter inwardly rectifying sodium-potassium current (IH)-6 These observations show that M-current inhibition by wortmannin has two pharmacological profiles. One is calcium-dependent and occurs at lower concentrations (0.1-1 LM), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3-10 IM), wortmannin has an additional, calciumindependent action, inhibiting the M-current by an unknown mechanism.

Experiments to Test the Role of Phosphatidylinositol 4,5-Bisphosphate in Neurotransmitter-Induced M-Channel Closure in Bullfrog Sympathetic Neurons

“…Our findings differ from those of a previous study in which wortmannin had a direct inhibitory effect on g M in BFSG neurons that was independent of the presence of agonist (Tokimasa et al, 1995). Differences in the nucleotide content of the internal solutions used in the two studies may provide an explanation for this disparity.…”

“…Although this is suggestive of a channel block mechanism, the effect of ML-7 on g M did not exhibit any obvious voltage or use dependence (our unpublished observations). It therefore may reflect a bona fide action on myosin light chain kinase, an enzyme that has been reported to affect g M (Akasu et al, 1993;Tokimasa et al, 1995). Despite this, ATP still suppressed the g M that persisted in the presence of ML-7; more importantly, the time course of recovery of the response was changed little (Fig.…”

Regulation of M-Type Potassium Current by Intracellular Nucleotide Phosphates

“…Several previous studies have suggested that I M is regulated by phosphorylation catalyzed by myosin light chain kinase. Application of myosin light chain kinase inhibitors decreases I M , whereas intracellular perfusion of catalytic subunits of myosin light chain kinase enhances I M (Akasu et al, 1993;Tokimasa et al, 1995). The result presented here, that ATP is required to prevent the rundown of I M , extends these findings to suggest that myosin light chain kinase-catalyzed phosphorylation events not only alter the amplitude of I M but may be required for the maintenance of I M .…”

Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons

“…Considering that inhibitors of MLCK may cause off‐target effects on action potential firings and voltage‐gated Ca 2+ channels (Tokimasa et al . ; Richards et al . ; Tokuoka and Goda ), we next studied effects of genetic down‐regulation of endogenous MLCK.…”