Alternative splicing determines sensitivity of murine calcium-activated potassium channels to glucocorticoids.
1. Large-conductance Ca(2+)- and voltage-activated potassium (BK) channels are important regulators of cellular excitability. Here, we present a patch-clamp electrophysiological analysis of splice-variant-specific regulation by the synthetic glucocorticoid dexamethasone (DEX) of BK channels consisting of cloned STREX or ZERO alpha-subunit variants expressed in human embryonic kidney (HEK 293) cells. 2. STREX channels in isolated membrane patches were inhibited by protein kinase A (PKA) and this was blocked on pre-treatment of intact cells with DEX (100 nM) for 2 h. 3. The effect of DEX required the synthesis of new mRNA and protein. Furthermore, it required protein phosphatase 2A (PP2A)-like activity intimately associated with the channels, as it was blocked by 10 nM okadaic acid but not by the specific protein phosphatase-1 inhibitor peptide PPI-2. 4. ZERO variant channels that lack the STREX insert were activated by PKA but were not influenced by DEX. ZERO channels containing a mutant STREX domain (S4(STREX)A) were also activated by PKA. Importantly, DEX blocked PKA activation of S4(STREX)A channels in a PP2A-dependent manner. 5. Taken together, the STREX domain is crucial for glucocorticoid regulation of BK channels through a PP2A-type enzyme. Moreover, glucocorticoids appear to induce a generic set of proteins in different types of cells, the actions of which depend on the expression of cell-specific targets.
Tian
L
L
Membrane Biology Group and MRC "Membrane and Adapter Protein" COOP, Section of Biomedical Sciences, University of Edinburgh Medical School, Hugh Robson Building, Edinburgh EH8 9XD, UK.
Hammond
M S
MS
Florance
H
H
Antoni
F A
FA
Shipston
M J
MJ
D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D003907:Dexamethasone / Q000494:pharmacology*; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000502:physiology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects* / Q000235:genetics*; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000378:metabolism; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D011965:Receptors, Glucocorticoid / Q000378:metabolism
D005938:Glucocorticoids; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D020033:Protein Isoforms; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2