Molecular and cellular correlates in Kv channel clustering: entropy-based regulation of cluster ion channel density.

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.

Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex.

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.

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

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.