Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts.

Katarina Ilic,Dirk Montag,Mario Stojanović,Ronald L Schnaar,Marija Heffer,Kristina Mlinac-Jerkovic,Marko Rožman,Željka Vukelić,Xiao Lin,Rodrigo Herrera-Molina,Ayse Malci,Svjetlana Kalanj-Bognar,Borna Puljko

doi:10.3390/ijms222413590

Abstract

The recent identification of plasma membrane (Ca2+)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis.

Highlights

The plasma membrane (PM) separates the intra- and extra-cellular environments.Positioning and function of membrane proteins in the PM is strongly influenced by dynamically changing lipid composition and interactions
In order to evaluate the effect of ganglioside composition on the exact submembrane localization of Neuroplastins and plasma membrane (Ca2+)-ATPase (PMCA), i.e., their abundance in lipid rafts (LR) and the bulk membrane, we performed lipid raft analysis isolation and Western blotting analysis of Nps and PMCAs expression in individual membrane fractions
In mice lacking complex gangliosides, Np65 is less enriched in LR (54%) and more dispersed in the bulk membrane (46%) so that the difference in LR and nLR localization is no longer significantly different (Supplementary Table S2)

Summary

Introduction

Positioning and function of membrane proteins in the PM is strongly influenced by dynamically changing lipid composition and interactions. The PM is not a simple nor a homogeneous milieu, but it is a highly dynamic structure resembling a patchwork of lipid raft microdomains with distinct composition and chemical properties. These nanometerscale submembrane compartments are enriched with (glyco)sphingolipids, cholesterol, and a specific subset of transmembrane proteins [1,2]. One family of transmembrane proteins for which fine-tuning of localization and activity is associated with lipid rafts is the Plasma Membrane (Ca2+ )-ATPase (PMCA) family.

Methods

Results

Conclusion