cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.
The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.
Nuwer
Megan O
MO
Program in Neuroscience, The State University of New York, Buffalo, NY 14214, USA.
Picchione
Kelly E
KE
Bhattacharjee
Arin
A
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019703:Calcineurin / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008957:Models, Genetic; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000235:genetics; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D000081033:Potassium Channels, Sodium-Activated; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D014162:Transfection
D002227:Carbazoles; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D000081033:Potassium Channels, Sodium-Activated; D011758:Pyrroles; C000626167:kcnt1 protein, rat; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C057416:KT 5720; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin