Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K channel modulates its interaction with scaffold proteins

Department of Life Sciences and Zlotowski Center for Neurosciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2007; 104(32):13022-7. DOI: 10.1073/pnas.0704059104
Source: PubMed


The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a "fishing rod" molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed.

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Available from: Uri Abdu, May 21, 2014
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    • "This is the first study to examine peculiarities of intrinsic disorder propensity in the large conductance calcium-activated K+ channel and its BKAPs. Several recent studies explored IDPRs in other channel types or their subunits, including the K+ voltage-gated channel [16], [64], the β-subunit of BK [65], and the NMDA receptor [66]. These IDPRs were defined mechanistically in relation to channel activity or interactions with cytoskeletal proteins. "
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    ABSTRACT: The large-conductance Ca2+-activated K+ (BK) channel is broadly expressed in various mammalian cells and tissues such as neurons, skeletal and smooth muscles, exocrine cells, and sensory cells of the inner ear. Previous studies suggest that BK channels are promiscuous binders involved in a multitude of protein-protein interactions. To gain a better understanding of the potential mechanisms underlying BK interactions, we analyzed the abundance, distribution, and potential mechanisms of intrinsic disorder in 27 BK channel variants from mouse cochlea, 104 previously reported BK-associated proteins (BKAPS) from cytoplasmic and membrane/cytoskeletal regions, plus BK β- and γ-subunits. Disorder was evaluated using the MFDp algorithm, which is a consensus-based predictor that provides a strong and competitive predictive quality and PONDR, which can determine long intrinsically disordered regions (IDRs). Disorder-based binding sites or molecular recognition features (MoRFs) were found using MoRFpred and ANCHOR. BKAP functions were categorized based on Gene Ontology (GO) terms. The analyses revealed that the BK variants contain a number of IDRs. Intrinsic disorder is also common in BKAPs, of which ∼5% are completely disordered. However, intrinsic disorder is very differently distributed within BK and its partners. Approximately 65% of the disordered segments in BK channels are long (IDRs) (>50 residues), whereas >60% of the disordered segments in BKAPs are short IDRs that range in length from 4 to 30 residues. Both α and γ subunits showed various amounts of disorder as did hub proteins of the BK interactome. Our analyses suggest that intrinsic disorder is important for the function of BK and its BKAPs. Long IDRs in BK are engaged in protein-protein and protein-ligand interactions, contain multiple post-translational modification sites, and are subjected to alternative splicing. The disordered structure of BK and its BKAPs suggests one of the underlying mechanisms of their interaction.
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    • "The column was calibrated with RNase A (13.7 kDa, 16.4 Å; where 1 Å=0.1 nM), chymotrypsinogen A (25.7 kDa, 20.9 Å), ovalbumin (44 kDa, 30.5 Å) and BSA (66.2 kDa, 35.5 Å). The results were analysed according to the method described previously [18]. "
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    ABSTRACT: Inteins are intervening protein sequences that undergo self-excision from a precursor protein with the concomitant ligation of the flanking polypeptides. Split inteins are expressed in two separated halves, and the recognition and association of two halves are the first crucial step for initiating trans-splicing. In the present study, we carried out the structural and thermodynamic analysis on the interaction of two halves of DnaE split intein from Synechocystis sp. PCC6803. Both isolated halves (IN and IC) are disordered and undergo conformational transition from disorder to order upon association. ITC (isothermal titration calorimetry) reveals that the highly favourable enthalpy change drives the association of the two halves, overcoming the unfavourable entropy change. The high flexibility of two fragments and the marked thermodynamic preference provide a robust association for the formation of the well-folded IN/IC complex, which is the basis for reconstituting the trans-splicing activity of DnaE split intein.
    Full-text · Article · Jun 2012 · Bioscience Reports
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    • "Shortening the KCNE1 proximal C-terminus may cause the tail to be more rigid and restrict its conformational states thereby increasing its association strength with Kv7.1. A similar mechanism seems to apply in the case of the intrinsically disordered Shaker channel C-terminus (Magidovich et al, 2007). Taken together, this study reveals the specific interaction between the KCNE1 C-terminus and the Kv7.1 dimeric coiledcoil helix C, thereby providing a simple means to guide assembly of the I KS channel complex. "
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    ABSTRACT: Voltage-gated K(+) channels co-assemble with auxiliary beta subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 beta subunits generates the repolarizing K(+) current I(KS). However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of I(KS) channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for I(KS) channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K(+) currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.
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