Dataset Information

Dynamic Distribution of ASIC1a Channels and Other Proteins within Cells Detected through Fractionation.

ABSTRACT: Proteins in eukaryotic cells reside in different cell compartments. Many studies require the specific localization of proteins and the detection of any dynamic changes in intracellular protein distribution. There are several methods available for this purpose that rely on the fractionation of the different cell compartments. Fractionation protocols have evolved since the first use of a centrifuge to isolate organelles. In this study, we described a simple method that involves the use of a tabletop centrifuge and different detergents to obtain cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins and identify the proteins in each fraction. This method serves to identify transmembrane proteins such as channel subunits as well as PM-embedded or weakly associated proteins. This protocol uses a minute amount of cell material and typical equipment present in laboratories, and it takes approximately 3 h. The process was validated using endogenous and exogenous proteins expressed in the HEK293T cell line that were targeted to each compartment. Using a specific stimulus as a trigger, we showed and quantified the shuttling of a protein channel (ASIC1a, acid sensing ion channel) from the MO fraction to the PM fraction and the shuttling of a kinase from a cytosolic location to a nuclear location.

SUBMITTER: Salinas Castellanos LC

PROVIDER: S-EPMC9027401 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Dynamic Distribution of ASIC1a Channels and Other Proteins within Cells Detected through Fractionation.

Salinas Castellanos Libia Catalina LC Gatto Rodolfo Gabriel RG Menchón Silvia Adriana SA Blaustein Matías M Uchitel Osvaldo Daniel OD Weissmann Carina C

Membranes 20220331 4

Proteins in eukaryotic cells reside in different cell compartments. Many studies require the specific localization of proteins and the detection of any dynamic changes in intracellular protein distribution. There are several methods available for this purpose that rely on the fractionation of the different cell compartments. Fractionation protocols have evolved since the first use of a centrifuge to isolate organelles. In this study, we described a simple method that involves the use of a tablet ...[more]

PMID: 35448360

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Project description:Trimeric acid-sensing ion channels (ASICs) contribute to neuronal signaling by converting extracellular acidification into excitatory sodium currents. Previous work with homomeric ASIC1a implicates conserved leucine (L7') and consecutive glycine-alanine-serine (GAS belt) residues near the middle, and conserved negatively charged (E18') residues at the bottom of the pore in ion permeation and/or selectivity. However, a conserved mechanism of ion selectivity throughout the ASIC family has not been established. We therefore explored the molecular determinants of ion selectivity in heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels using site-directed mutagenesis, electrophysiology, and molecular dynamics free energy simulations. Similar to ASIC1a, E18' residues create an energetic preference for sodium ions at the lower end of the pore in ASIC2a-containing channels. However, and in contrast to ASIC1a homomers, ion permeation through ASIC2a-containing channels is not determined by L7' side chains in the upper part of the channel. This may be, in part, due to ASIC2a-specific negatively charged residues (E59 and E62) that lower the energy of ions in the upper pore, thus making the GAS belt more important for selectivity. This is confirmed by experiments showing that the L7'A mutation has no effect in ASIC2a, in contrast to ASIC1a, where it eliminated selectivity. ASIC2a triple mutants eliminating both L7' and upper charges did not lead to large changes in selectivity, suggesting a different role for L7' in ASIC2a compared with ASIC1a channels. In contrast, we observed measurable changes in ion selectivity in ASIC2a-containing channels with GAS belt mutations. Our results suggest that ion conduction and selectivity in the upper part of the ASIC pore may differ between subtypes, whereas the essential role of E18' in ion selectivity is conserved. Furthermore, we demonstrate that heteromeric channels containing mutations in only one of two ASIC subtypes provide a means of functionally testing mutations that render homomeric channels nonfunctional.

Dataset Information

Dynamic Distribution of ASIC1a Channels and Other Proteins within Cells Detected through Fractionation.

Publications

Dynamic Distribution of ASIC1a Channels and Other Proteins within Cells Detected through Fractionation.

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