Small Conductance Ca2+-activated K+ Channels and Calmodulin: CELL SURFACE EXPRESSION AND GATING

Wei-Sheng Lee,Thu Jennifer Ngo-Anh,Andrew Bruening-Wright,James Maylie,John P Adelman

doi:10.1074/jbc.m302091200

Wei-Sheng Lee, Thu Jennifer Ngo-Anh + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m302091200

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Abstract

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.

Highlights

Small conductance Ca2؉-activated K؉ channels (SK channels) are heteromeric complexes of pore-forming ␣ subunits and constitutively bound calmodulin (CaM)
The results presented here show that Ca2ϩ-independent interactions between the CaM binding domain (CaMBD) and CaM are essential for cell surface expression and that the constitutive binding between the pore-forming ␣ subunits of SK channels and CaM is not required for channel gating
The purified CaMBD R464E/K467E peptide failed to retain purified CaM in pull-down assays in the absence of Ca2ϩ [14]. These results show that the CaMBD R464E/K467E is different from wild type in its ability to retain CaM, but they do not distinguish between a complete lack of binding or a weakened affinity

Summary

Introduction

Small conductance Ca2؉-activated K؉ channels (SK channels) are heteromeric complexes of pore-forming ␣ subunits and constitutively bound calmodulin (CaM). 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. Structure-function studies are consistent with this model and have shown that the interaction occurs at the CaM binding domain (CaMBD), a highly conserved stretch of 92 amino acids residing in the proximal region of the intracellular C terminus of the ␣ subunits [14, 15]. Residues in the linker domain and the C-lobe maintain Ca2ϩ-independent interactions, including salt bridges between Arg-464 and Lys-467 on the CaMBD and Glu-84 and Glu-87 on CaM.

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