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

Abstract

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.channel clustering ͉ intrinsically disordered ͉ PSD-95

Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism

“…Our results can be rationalized by considering the entropic contribution of the binding reaction; shorter chains lose less entropy upon binding than do longer chains as the configurational entropy of the latter is restricted to a greater extent upon binding 13,28 . Our findings thus argue that the A and B tail splice variants are pure entropic (random) chains, the lengths of which determine affinity for the scaffold protein partner.…”

“…We next examined whether the differences in the affinities of Kv channel natural variants to PSD-95, seen at the molecular level, are also reflected in the cellular context. Channel cell surface expression was first examined by transfecting embryonic Drosophila Schneider cells to express the isolated Shaker A or B tails fused to the CD8 membrane-targeting sequence, either alone or along with a PSD-95-GFP fusion protein, as described before 13 . Cell surface expression was assessed by confocal microscopy.…”

“…The PSD-95 protein used in the current study is the Drosophila S97 variant. Cloning of the C-terminal segment of the Shaker A channel variant (DShA-C) into the His-tagencoding pHis parallel vector was performed as described previously 13 , as was cloning of the second PSD-95 PDZ domain (PDZ 2 ) into the pET28a plasmid. The 144 amino-acid-long wild-type ShB-C tail was shortened to lengths of 104 (513-553) and 61 (D513-596) residues using looping out a Quick change mutagenesis kit (Stratagene).…”

“…Instead, as demonstrated for the prototypical Shaker B Kv channel variant, the interaction between the two proteins is mediated by a long and flexible intrinsically disordered segment of the channel tail carrying a short linear sequence motif at its tip able to bind the globular PDZ domains of PSD-95 (refs 12,13; see also refs 14,15 for reviews on intrinsically disordered proteins and their functions). Perturbations that change the intrinsically disordered nature of the Shaker B channel tail, in particular gross modifications that shorten the entire tail or affect its flexibility, or disruption of the canonical PDZ-binding motif were shown to affect scaffold protein binding 13 . Based on these observations, a fishing rod-like description of the interaction of the Kv channel and scaffold protein has been suggested, whereby disorder in the C-terminal tail of the channel would provide the orientational freedom needed for searching and finding the PSD-95 scaffold protein partner 13 .…”

“…Perturbations that change the intrinsically disordered nature of the Shaker B channel tail, in particular gross modifications that shorten the entire tail or affect its flexibility, or disruption of the canonical PDZ-binding motif were shown to affect scaffold protein binding 13 . Based on these observations, a fishing rod-like description of the interaction of the Kv channel and scaffold protein has been suggested, whereby disorder in the C-terminal tail of the channel would provide the orientational freedom needed for searching and finding the PSD-95 scaffold protein partner 13 . Given the stoichiometry of the interaction of the two proteins, dictated by the three consecutive PDZ interaction modules of PSD-95 and the four C-terminal tails of the oligomeric Kv channel, channel clustering results 16 .…”