Small conductance Ca2+-activated K+ channels and calmodulin: cell surface expression and gating.
Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming alpha subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.
Department of Obstetrics and Gynecology and Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, USA.
Wei-Sheng
WS
Lee
Thu Jennifer
TJ
Ngo-Anh
Andrew
A
Bruening-Wright
James
J
Maylie
John P
JP
Adelman
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009154:Mutation; D015221:Potassium Channels / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D015220:Calcium Channels; D002147:Calmodulin; D015221:Potassium Channels; D012333:RNA, Messenger; D002118:Calcium