Membrane targeting of calcium-pumping atpase

Alternative splicing affects the membrane localization of the plasma membrane calcium pump (PMCA) isoform 2b. Full-length PMCA2w/b, PMCA2x/b, and PMCA2z/b (constructs schematically shown on top) were expressed in polarized MDCK (dog kidney epithelial) cells, and the localization of the pumps was studied by confocal fluorescence microscopy. PMCA2 was detected with an isoform-specific antibody followed by an Alexa-488 labeled secondary antibody (green). Nuclei were stained with DAPI. PMCA2w/b shows apical localization in addition to basal and lateral staining (left panel), whereas PMCA2x/b and PMCA2z/b are confined to the basal and lateral membranes (middle and right panel). Corresponding x:z sections are shown below the en face views. Arrows indicate the height at which the en face sections were captured. Bar, 20 µm. For details, see Chicka, M.C. and Strehler, E.E., J. Biol. Chem. 278:18464-18470 (2003).

Plasma membrane calcium ATPases (PMCAs) pump Ca2+ out of all animal cells. As components of the Ca2+ signaling “toolbox”, the PMCAs are localized in distinct plasma membrane domains. The importance of proper membrane targeting is illustrated by diseases characterized by the absence of specific PMCA isoforms in the membrane, such as deafness caused by a lack of PMCA2 in cochlear hair cell stereocilia. The long-term goal of this project is to understand the mechanism and functional impact of specific membrane targeting of the PMCAs. Specifically, we wish to identify the apical targeting elements in PMCA2 splice variants; to determine if and how PDZ protein interactions stabilize PMCA2 in the apical membrane; and to determine the functional effect of specific localization of PMCA2 splice variants on trans-epithelial Ca2+ flux and global Ca2+ signaling in polarized cells. The studies will involve confocal fluorescence microscopy and two-hybrid interaction analyses to identify specific targeting elements of PMCA2 splice variants. Fluorescence recovery after photobleaching and half-life studies will be employed to analyze the membrane dynamics of PMCA2 isoforms. Functional studies involve trans-epithelial Ca2+ flux measurements and ratiometric Ca2+ imaging in polarized MDCK cells. This work will explore the novel concept that the physiological role of the PMCAs is tightly linked to their precise localization in the membrane.

Relevance to public health: PMCA2 is abundant in neurons and is specifically localized within these cells. Although intracellular Ca2+ movements are an essential part of neurotransduction, the relative roles of the channels and pumps which control Ca2+ movements are not well understood. These studies will help us understand the mechanisms by which PMCA2 contributes to control of Ca2+. Understanding these mechanisms will help us understand and fight diseases caused by defects in local calcium regulation, such as hearing loss and neuronal degeneration in aging.