RESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Activación del Canal Iónico , Densidad Postsináptica/metabolismo , Canales de Potasio de la Superfamilia Shaker/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Drosophila , Entropía , Humanos , Unión ProteicaRESUMEN
The fast inactivation and clustering functions of voltage-dependent potassium channels play fundamental roles in electrical signaling. Recent evidence suggests that both these distinct channel functions rely on intrinsically disordered N- and C-terminal cytoplasmic segments that function as entropic clocks to time channel inactivation or scaffold protein-mediated clustering, both relying on what can be described as a "ball and chain" binding mechanism. Although the mechanisms employed in each case are seemingly analogous, both were put forward based on bulky chain deletions and further exhibit differences in reaction order. These considerations raised the question of whether the molecular mechanisms underlying Kv channel fast inactivation and clustering are indeed analogous. By taking a "chain"-level chimeric channel approach involving long and short spliced inactivation or clustering "chain" variants of the Shaker Kv channel, we demonstrate the ability of native inactivation and clustering "chains" to substitute for each other in a length-dependent manner, as predicted by the "ball and chain" mechanism. Our results thus provide direct evidence arguing that the two completely unrelated Shaker Kv channel processes of fast inactivation and clustering indeed occur according to a similar molecular mechanism.
Asunto(s)
Canales de Potasio/metabolismo , Animales , Análisis por Conglomerados , Citoplasma/metabolismo , Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación del Canal Iónico/fisiología , Proteínas de la Membrana/metabolismo , Unión ProteicaRESUMEN
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.
Asunto(s)
Apoferritinas/metabolismo , Neoplasias Encefálicas/diagnóstico por imagen , Compuestos Férricos/metabolismo , Óxido Ferrosoférrico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Apoferritinas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Genes Reporteros , Ingeniería Genética , Imagen por Resonancia Magnética , Ratones , Modelos Moleculares , Trasplante de Neoplasias , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
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.
Asunto(s)
Entropía , Activación del Canal Iónico/fisiología , Modelos Biológicos , Canales de Potasio con Entrada de Voltaje/fisiología , Animales , Línea Celular , Potenciales de la Membrana/fisiología , Modelos Moleculares , Canales de Potasio con Entrada de Voltaje/química , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
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.