RESUMEN
Small-conductance calcium-activated potassium (SK) channels show a ubiquitous distribution on neurons, in both somatodendritic and axonal regions. SK channels are associated with neuronal activity regulating action potential frequency, dendritic excitability, and synaptic plasticity. Although the physiology of SK channels and the mechanisms that control their surface expression levels have been investigated extensively, little is known about what controls SK channel diffusion in the neuronal plasma membrane. This aspect is important, as the diffusion of SK channels at the surface may control their localization and proximity to calcium channels, hence increasing the likelihood of SK channel activation by calcium. In this study, we successfully investigated the diffusion of SK channels labeled with quantum dots on human embryonic kidney cells and dissociated hippocampal neurons by combining a single-particle tracking method with total internal reflection fluorescence microscopy. We observed that actin filaments interfere with SK mobility, decreasing their diffusion coefficient. We also found that during neuronal maturation, SK channel diffusion was gradually inhibited in somatodendritic compartments. Importantly, we observed that axon barriers formed at approximately days in vitro 6 and restricted the diffusion of SK channels on the axon initial segment (AIS). However, after neuron maturation, SK channels on the AIS were strongly immobilized, even after disruption of the actin network, suggesting that crowding may cause this effect. Altogether, our work provides insight into how SK channels diffuse on the neuronal plasma membrane and how actin and membrane crowding impacts SK channel diffusion.
Asunto(s)
Actinas , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Humanos , Actinas/metabolismo , Neuronas , Axones/metabolismo , Hipocampo/metabolismoRESUMEN
It was recently revealed via super-resolution microscopy experiments that the axon plasma membrane skeleton (APMS) comprises a series of periodically arranged azimuthal actin rings connected via longitudinal spectrin filaments forming an orthotropic network. The common perception is that APMS enhances structural stability of the axon but its impact on axon deformation is unknown. To investigate the response of the APMS to extension, we introduce a coarse-grain molecular dynamics model consisting of actin particles forming rings and chains of particles representing spectrin tetramers with repeats than can unfold. We observe that the shape of force-extension curve is initially linear and the force level depends on the extension rate. Even during the initial deformation stage, unfolding of spectrin repeats occurs, but the saw-tooth shape of the corresponding force-extension curve observed in the case of one spectrin tetramer does not appear in the case of the entire APMS. The reason is that spectrin unfolding is not synchronized across filaments during extension. If actin-spectrin associations remain intact, the force-extension response reaches a perfectly plastic region because of increased spectrin unfolding frequency. However, when actin-spectrin links dissociate, which can happen at moderate and high extension rates, APMS softens and the resistance force decreases linearly as the axon elongates until it reaches a point where the APMS is completely severed. Furthermore, when the ring-to-ring distance is maintained fixed under stretch, the resistance force relaxes exponentially as a function of time due to additional unfolding of spectrin tetramers following the Kelvin-Voigt representation of the Zener model.