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Inhibition by wortmannin of M-current in bullfrog sympathetic neurones
T Tokimasa1, M Ito, M A Simmons
1Department of Physiology, Kurume University School of Medicine, Japan.
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 (0.01-10 microM) 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 microM wortmannin. Wortmannin, 3 microM, 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 microM 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 microM) did not affect rapidly-inactivating A-type or delayed rectifier-type potassium currents not 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 microM), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3-10 microM), wortmannin has an additional, calcium-independent action, inhibiting the M-current by an unknown mechanism.
Insights
Wortmannin inhibits M-type potassium current in bullfrog neurons through a calcium-dependent mechanism at lower doses and a calcium-independent mechanism at higher doses, affecting myosin light chain kinase (MLCK).
Area of Science:
- Neuroscience
- Pharmacology
- Cell Physiology
Background:
- The M-type potassium current (IM) is crucial for neuronal excitability.
- Myosin light chain kinase (MLCK) plays a role in regulating ion channel function.
- Wortmannin is a known inhibitor of MLCK.
Purpose of the Study:
- To investigate the effects of wortmannin on the M-type potassium current in bullfrog sympathetic neurons.
- To elucidate the role of calcium and MLCK in wortmannin's action on M-current.
Main Methods:
- Whole-cell patch-clamp recordings were used to measure M-current amplitude.
- Bullfrog sympathetic neurons were pretreated with varying concentrations of wortmannin (0.01-10 microM).
- Internal solutions with different calcium concentrations (pCa 6, 7, 8) were employed.
Main Results:
- Wortmannin inhibited M-current in a dose-dependent manner, with significant inhibition at 3-10 microM.
- A calcium-dependent inhibition of M-current by wortmannin (0.1-1 microM) was observed, linked to MLCK inhibition.
- Higher wortmannin concentrations (3-10 microM) showed additional calcium-independent M-current inhibition.
- Other potassium currents (A-type, delayed rectifier) and sodium-potassium inward rectifier current (IH) were unaffected.
Conclusions:
- Wortmannin exhibits dual mechanisms for inhibiting M-current: calcium-dependent via MLCK inhibition at lower doses and calcium-independent at higher doses.
- These findings highlight the complex regulation of M-current by calcium and MLCK.
- Wortmannin serves as a valuable tool for dissecting the pharmacological properties of M-current.