Abstract
The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 A crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.
Highlights
The KCNQ channels represent a subfamily of voltage-gated Kϩ (Kv)3 channels, whose members (Kv7.1-5) are expressed in a wide variety of tissues
Our findings suggest that the KCNQ1 COOH terminus may be divided into two parts, where the membrane proximal half is important for functional expression, folding, and gating of the channel but not oligomerization, whereas the membrane distal half directs folding, oligomerization, partner specificity, and trafficking to the plasma membrane
The results indicate that the complex is best modeled as a tetramer with four subunits of KCNQ1 COOH terminus and four bound molecules of CaM (Fig. 1D)
Summary
PCR was used to engineer BamHI and NotI restriction sites onto the specific human KCNQ1 gene fragments. PCR product was ligated into doubly digested (BamHI and NotI) modified pETDuet vector. PCR was used to engineer EcoRI and NotI restriction sites onto an additional gene fragment (residues 504 – 622) This PCR product was ligated into doubly digested (EcoRI and NotI) modified pETDuet vector already contained the fragment encoding residues 352–386. GCN4-LI PCR product was ligated into doubly digested (EcoRI and NotI) modified pETDuet vector that already contained DNA encoding residues 352–594. For purification of ⌬ helix D, ⌬ helices C-D, and ⌬ loopCaM complexes, a Superdex-200 gel filtration column was employed instead of the Superose-6 gel filtration column Both KCNQ1 gene fragments (residues 535–572 or 535– 622, respectively) were amplified by PCR with primers containing BamHI and NotI sites in the 5Ј- and 3Ј-flanking regions, respectively.
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