A Helical Region in the C Terminus of Small-conductance Ca2+-activated K+ Channels Controls Assembly with Apo-calmodulin

Ralph Wissmann,Wolfgang Bildl,Heinz Neumann,Andre F Rivard,Nikolaj Klöcker,Dietmar Weitz,Uwe Schulte,John P Adelman,Detlef Bentrop,Bernd Fakler

doi:10.1074/jbc.m109240200

Ralph Wissmann, Wolfgang Bildl + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m109240200

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Abstract

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.

Highlights

Small conductance Ca2ϩ-activated potassium (SK)1 channels are responsible for the prolonged afterhyperpolarization that follows the action potential in many central neurons and is a major determinant of excitability and firing pattern [1,2,3,4]
Interactions Between CaMBD and ApoCaM—The Ca2ϩ-free association between CaMBD and CaM was examined in NMR experiments on the apo(CaMBD1⁄7CaM) complex reconstituted in solution
Our results show that the solution structure of the SK2 CaMBD consists of highly flexible N and C termini that flank a well ordered core region forming an unusually folded helical domain between Ala-423 (Ala-28 in CaMBD) and Asn-437 (Asn-42 in CaMBD)

Summary

Introduction

Small conductance Ca2ϩ-activated potassium (SK) channels are responsible for the prolonged afterhyperpolarization that follows the action potential in many central neurons and is a major determinant of excitability and firing pattern [1,2,3,4]. SK subunits share the prototypic membrane topology of voltagegated Kϩ (Kv) channels with six transmembrane domains flanked by cytoplasmic N and C termini (Fig. 1), their primary sequences do not contain any significant homology outside the P-loop As for their native counterparts, cloned SK channels are solely gated by [Ca2ϩ]i in the submicromolar range, independent of the transmembrane voltage (7, 10 –12). Analysis of Ca2ϩ-gating showed that the high affinity of SK channels for Ca2ϩ results from a property, unique among the large family of Kϩ channels: the SK ␣-subunits associate with the Ca2ϩ-binding protein calmodulin (CaM) [12] This association is constitutive, i.e. it occurs in the absence of Ca2ϩ.

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