Ion channel clustering at the post-synaptic density serves a fundamental role in action potential generation and transmission. Here, we show that interaction between the Shaker Kv channel and the PSD-95 scaffold protein underlying channel clustering is modulated by the length of the intrinsically disordered C terminal channel tail. We further show that this tail functions as an entropic clock that times PSD-95 binding. We thus propose a 'ball and chain' mechanism to explain Kv channel binding to scaffold proteins, analogous to the mechanism describing channel fast inactivation. The physiological relevance of this mechanism is demonstrated in that alternative splicing of the Shaker channel gene to produce variants of distinct tail lengths resulted in differential channel cell surface expression levels and clustering metrics that correlate with differences in affinity of the variants for PSD-95. We suggest that modulating channel clustering by specific spatial-temporal spliced variant targeting serves a fundamental role in nervous system development and tuning.

Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structurefunction analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cyclecoupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented. activation gate ͉ allosteric enzymes ͉ double-mutant cycles ͉ voltage-activated potassium channels ͉ non-additivity A llosteric regulation of protein function is often achieved by changes in protein conformation induced by the binding of substrate or other ligand molecules (1). In multisubunit proteins, such conformational changes may give rise to cooperativity in ligand binding. Several mechanistic models, in particular the Monod-Wymann-Changeux (MWC) model (2) and the Koshland-Némethy-Filmer (KNF) model (3), were developed in the 1960s to describe cooperativity in ligand binding. In both models, cooperativity is explained by binding-induced conformational changes in the subunits of the protein that may be concerted (MWC), sequential (KNF), or a combination of both (4). No matter the type of conformational change, cooperativity in ligand binding in all allosteric models is manifested by changes in intersubunit interactions along the ligation pathway of the protein (1).Despite the general acceptance of intersubunit interactions playing a fundamental role in determining the magnitude of cooperativity in ligand binding by an allosteric enzyme, structure-function analysis of such proteins throu...

Edited by Wilhelm JustKeywords: 'Ball and chain' Clustering Entropic chain Inactivation Intrinsic disorder Scaffold protein PSD-95 Voltage-dependent potassium channel a b s t r a c t Electrical signaling in the nervous system relies on action potential generation, propagation and transmission. Such processes are dynamic in nature and rely on precisely timed events associated with voltage-dependent ion channel conformational transitions between their primary open, closed and inactivated states and clustering at unique membrane sites. In voltage-dependent potassium (Kv) channels, fast inactivation and clustering processes rely on entropic clock chains as described by 'ball and chain' mechanisms, suggesting important roles for such chains in electrical signaling. Here, we consider evidence supporting the proposed 'ball and chain' mechanisms for Kv channel fast inactivation and clustering associated with intrinsically disordered N-and C-terminal regions of the protein, respectively. Based on this comparison, we delineate the requirements that argue for such a process and establish the thermodynamic signature of a 'ball and chain' mechanism. Finally, we demonstrate how 'chain'-level alternative splicing of the Kv channel gene modulates the entropic clock-based 'ball and chain' inactivation and clustering channel functions underlying changes in electrical signaling. As such, the Kv channel model system exemplifies how linkage between alternative splicing and intrinsic disorder enables functional diversity.

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The 24a Sunday,

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