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State-dependent modulation of mSlo, a cloned calcium-dependent potassium channel
M Müller1, D Madan, I B Levitan
1Biochemistry Department, Brandeis University, Waltham, MA 02254, USA.
Abstract:
The mouse slopoke calcium-dependent potassium channel (mSlo) has been expressed heterologously in COS cells, and incorporated from COS cell membranes into artificial phospholipid bilayers. Under control conditions, the channel is not modulated by ATP. However, when mSlo is treated first with the calcium-dependent potassium channel opener NS004, which itself increases the open probability of the channel, subsequent addition of ATP causes a large further increase in channel open probability. An increase in channel activity is not by itself sufficient to confer sensitivity to ATP, because ATP does not modulate channels whose open probability has been increased by elevated calcium or depolarized voltage. The ATP analog AMP-PNP has only minimal effects on channel activity after treatment with NS004, suggesting that hydrolysis of the ATP is required for its action on mSlo. A peptide inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII) blocks the modulation of mSlo by ATP, whereas peptide inhibitors of other serine/threonine protein kinases are without effect. The results are consistent with a state-dependent modulation of mSlo by ATP, possibly via phosphorylation.
Insights
The mouse slo potassium channel (mSlo) activity is modulated by ATP only after treatment with specific openers like NS004. This ATP modulation likely involves calcium/calmodulin-dependent protein kinase II (CaMKII) and requires ATP hydrolysis.
Area of Science:
- Biophysics
- Molecular Biology
- Ion Channel Physiology
Background:
- The mouse slo potassium channel (mSlo) is a calcium-dependent ion channel.
- Understanding mSlo regulation is crucial for cellular excitability studies.
- ATP is a key cellular energy source that can regulate protein function.
Purpose of the Study:
- To investigate the modulatory effect of ATP on the mouse slo calcium-dependent potassium channel (mSlo).
- To determine the conditions under which ATP influences mSlo channel activity.
- To elucidate the potential mechanisms underlying ATP-mediated mSlo modulation.
Main Methods:
- Heterologous expression of mSlo in COS cells.
- Incorporation of mSlo into artificial phospholipid bilayers.
- Electrophysiological recordings to measure channel open probability.
- Application of ATP, ATP analogs (AMP-PNP), channel openers (NS004), and kinase inhibitors (CaMKII inhibitors).
Main Results:
- mSlo channels were not modulated by ATP under control conditions.
- Treatment with the opener NS004 rendered mSlo channels sensitive to ATP, significantly increasing their open probability.
- ATP did not modulate mSlo channels activated by elevated calcium or depolarized voltage.
- ATP analog AMP-PNP showed minimal effects, suggesting ATP hydrolysis is required.
- A CaMKII inhibitor blocked ATP's modulatory effect, while other kinase inhibitors had no effect.
Conclusions:
- ATP modulates mSlo channel activity in a state-dependent manner.
- The modulation appears to be mediated by phosphorylation, likely involving CaMKII.
- ATP hydrolysis is necessary for this modulatory effect.
- This suggests a complex regulatory mechanism for mSlo channels involving ATP and specific cellular signaling pathways.