Biochemical analysis of ASIC1a channel distribution within HEK293 cells

CASTELLANOS, LIBIA C SALINAS; OSVALDO D. UCHITEL; CARINA WEISSMANN

CABA

Congreso; SAN 2022; 2022

SAN

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. he 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 established a simple and quick method that involves the use of HEK293 cell lines exogenously expressing the protein of interest, a tabletop centrifuge and different detergents to obtain cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins to identify the proteins in each fraction. Using specific stimuli as a trigger, we describe and quantify the shuttling of ASIC1a channels, as well as the shuttling of a kinase from different compartments within cells.This work shows the mechanisms by which different conditions could determine ASIC1a channels (and other proteins) altered distribution, and thus its function be modulated in tissue acidosis, such as ischemic stroke, epileptic seizures, chronic pain, and neurodegenerative diseases.