Cellular and Molecular Neurobiology
Author: LIBIA CATALINA SALINAS CASTELLANOS | email: libia_catalina53@hotmail.com
Libia Catalina Salinas Castellanos 1°, Osvaldo Daniel Uchitel 1°, Carina Weissmann 1°
1° Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE – CONICET – UBA)
Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases as well as pain conditions. Classically, ASICs are described as transiently activated by a reduced pH, followed by desensitization; the activation allows sodium influx, and in the case of ASIC1a-composed channels, also calcium to some degree. The function of ASICs depends on the number of channels on the cell surface. Thus, the dynamic control of surface ASICs under normal and pathological conditions is highly relevant. In this work we present a simple and quick fractionation method we established that involves the use of HEK293 cell lines exogenously expressing the protein of interest, a tabletop centrifuge and different detergents. In hours, different cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins are obtained allowing for the identification of proteins in each by western blot. Using specific stimuli as a trigger, and fractionation, we describe and quantify the shuttling of ASIC1a channels, as well as the shuttling of a kinase, between cell compartments. This work shows ASIC1a channels (and other proteins) altered distribution in different conditions that in turn can lead to changes in currents that might be relevant in tissue acidosis as present in ischemic stroke, epileptic seizures, chronic pain, and neurodegenerative diseases.