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.

Scaffold protein-mediated ion channel clustering at unique membrane sites is important for electrical signaling. Yet, the mechanism(s) by which scaffold protein-ion channel interactions lead to channel clustering or how cluster ion channel density is regulated is mostly not known. the voltage-activated potassium channel (Kv) represents an excellent model to address these questions as the mechanism underlying its interaction with the post-synaptic density 95 (PSD-95) scaffold protein is known to be controlled by the length of the extended 'ball and chain' sequence comprising the C-terminal channel region. Here, using sub-diffraction high-resolution imaging microscopy, we show that Kv channel 'chain' length regulates Kv channel density with a 'bell'-shaped dependence, reflecting a balance between thermodynamic considerations controlling 'chain' recruitment by PSD-95 and steric hindrance due to the spatial proximity of multiple channel molecules. our results thus reveal an entropy-based mode of channel cluster density regulation that mirrors the entropy-based regulation of the Kv channel-PSD-95 interaction. The implications of these findings for electrical signaling are discussed. Action potential generation, propagation and the evoked synaptic potential all rely on precisely timed events associated with activation and inactivation gating transitions of voltage-dependent Na + and K + channels, clustered in multiple copies at unique membrane sites, such as the initial segment of an axon, nodes of Ranvier, pre-synaptic terminals or at the post-synaptic density (PSD) 1-3. Changes in either ionic current shape or density, reflecting changes in temporal and spatial dimensions, respectively, affect action potential shape and frequency and may lead a neuron to change its mode of firing 4-9. Despite emerging evidence attesting to the importance of ion channel density for efficient electrical signaling 7 and information encoding 7-9 , little is currently known of the clustering process itself or its regulation. It is, however, clear that ion channel clustering is an active process, involving the interaction of a channel with a specific member of one of several scaffold protein families. For example, Nav channel clustering at nodes of Ranvier is mediated by interaction of the channel with the ankyrin G scaffold protein 10,11. Kv channel clustering at the PSD of excitatory synapses (of Drosophila melanogaster but not of mammalians), on the other hand, is mediated by binding to the PSD-95 synapse-associated scaffold protein 12-15. Yet, the mechanism by which these elementary binding events lead to the clustering of ion channel molecules in a restricted area of the membrane remains unclear. Furthermore, it is also generally not known if and how ion channel membrane density is regulated in the spatial and/or temporal dimensions. In the absence of a molecular mechanism describing the channel protein-scaffold protein interaction, bridging this molecularcellular gap to understand ion channel clustering has proven challenging. The pr...

No abstract

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.

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene – ferritin-M6A – in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.