title: PKA reduces the rat and human KCa3.1 current, CaM binding, and Ca2+ signaling, which requires Ser332/334 in the CaM-binding C terminus.
abstract: The Ca(2+)-dependent K(+) channel, KCa3.1 (KCNN4/IK/SK4), is widely expressed and contributes to cell functions that include volume regulation, migration, membrane potential, and excitability. KCa3.1 is now considered a therapeutic target for several diseases, including CNS disorders involving microglial activation; thus, we need to understand how KCa3.1 function is regulated. KCa3.1 gating and trafficking require calmodulin binding to the two ends of the CaM-binding domain (CaMBD), which also contains three conserved sites for Ser/Thr kinases. Although cAMP protein kinase (PKA) signaling is important in many cells that use KCa3.1, reports of channel regulation by PKA are inconsistent. We first compared regulation by PKA of native rat KCa3.1 channels in microglia (and the microglia cell line, MLS-9) with human KCa3.1 expressed in HEK293 cells. In all three cells, PKA activation with Sp-8-Br-cAMPS decreased the current, and this was prevented by the PKA inhibitor, PKI14-22. Inhibiting PKA with Rp-8-Br-cAMPS increased the current in microglia. Mutating the single PKA site (S334A) in human KCa3.1 abolished the PKA-dependent regulation. CaM-affinity chromatography showed that CaM binding to KCa3.1 was decreased by PKA-dependent phosphorylation of S334, and this regulation was absent in the S334A mutant. Single-channel analysis showed that PKA decreased the open probability in wild-type but not S334A mutant channels. The same decrease in current for native and wild-type expressed KCa3.1 channels (but not S334A) occurred when PKA was activated through the adenosine A2a receptor. Finally, by decreasing the KCa3.1 current, PKA activation reduced Ca(2+)-release-activated Ca(2+) entry following activation of metabotropic purinergic receptors in microglia.
authors.affiliation: Genes and Development Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8, Canada and Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Raymond
authors.initials: R
authors.lastname: Wong
authors.affiliation: Genes and Development Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8, Canada and Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada schlicht@uhnres.utoronto.ca.
authors.forename: Lyanne C
authors.identifier: http://orcid.org/0000-0002-8122-027X
authors.initials: LC
authors.lastname: Schlichter
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism*
chemical_list: D001562:Benzimidazoles; D002120:Calcium Channel Agonists; D002147:Calmodulin; D004791:Enzyme Inhibitors; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; C491575:Kcnn4 protein, rat; D012694:Serine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C102635:1-ethyl-2-benzimidazolinone; D002118:Calcium
keywords: SK channel regulation; adenosine A2a receptor; calmodulin binding; microglia ion channels; mutation analysis; protein kinase A