Membrane Targeting and Function of Plasma Membrane Ca2+ (PMCA) Pumps
The Na+/H+-exchanger regulatory factor-2 (NHERF2) anchors the plasma membrane calcium pump isoform PMCA2w/b to the apical membrane in polarized MDCK cells. Confocal fluorescence images are shown of the apical membrane of MDCK cells co-expressing GFP-PMCA2w/b (green, A1) and NHERF2 (red, A2), and stained for actin (blue, A3). Panel A4 is a merged image, where white indicates overlap of the green, red and blue staining.
Illustration B shows anchorage of PMCA2w/b to the apical actin cytoskeleton by NHERF2 via ezrin. PMCA2w/b binds via its C-terminal ETSL sequence to the PDZ protein NHERF2, which interacts through its ERM domain with ezrin.
Source: Padanyi R, et al. Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells. The Journal of Biological Chemistry. 2010;285:31704.
Specific mutations of plasma membrane Ca2+ pump isoform 2 (PMCA2) lead to hearing impairment, ataxia and other neurological problems in mice. They have also recently been linked to hearing loss in humans.
Dr. Strehler's laboratory has shown that a unique splice form of PMCA2 is targeted exclusively to the apical membrane of inner ear hair cells and polarized epithelial cells, leading the lab to ask:
- What sequence and structural features provide the specific apical targeting signal in PMCA2?
- What other proteins are involved in PMCA2 targeting?
- How do differently targeted PMCA2 variants control local Ca2+ regulation in submembrane Ca2+ microdomains?
Using polarized Madin-Darby canine kidney (MDCK) cells as a model system, the laboratory is addressing these questions by confocal fluorescence microscopy and various biochemical and cellular assays. Additionally, the lab is developing novel targeted calcium sensors to probe the in situ function of differently localized calcium pump isoforms.
These studies are performed in close collaboration with the laboratory of Agnes Enyedi, Ph.D., in Budapest, Hungary.
Public health relevance
Stable expression and function of PMCA2w/b in MDCK cells. Image A (top) shows MDCK cells stably expressing PMCA2w/b co-stained with antibodies against PMCA2 (green), Na+/K+-ATPase (blue) and ezrin (red). A substantial overlap of the fluorescence signals for PMCA2 and ezrin is observed on the apical side (merged image on bottom). Cells were analyzed by confocal laser scanning microscopy with vertical z-scans.
Image B (bottom) shows the time course of Ca2+ transients measured in empty vector control or PMCA2w/b expressing MDCK cells transfected with the genetically encoded Ca2+ indicator GCaMP2. The arrow indicates administration of 100 micrometer ATP to elicit a Ca2+ spike. Twenty to 30 cells were measured in a single experiment and the experimental data were fitted using GraphPad Prism4 software. This experiment demonstrates that expression of PMCA2w/b dramatically shortens the duration of the Ca2+ signal compared to control cells that express mainly PMCA4b.
Source: Antalffy G, et al. Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells. Biochemical and Biophysical Research Communications. 2011;410:322.
PMCAs are essential for the regulation of intracellular Ca2+ levels. PMCA isoforms with specific functions are enriched in different membrane compartments, such as at neuronal synapses or in the apical membrane of cochlear hair cells and mammary epithelial cells.
Although localized Ca2+ movements are an integral part of processes such as neurotransduction, the relative roles of the channels and pumps that control Ca2+ movements are not well understood.
These studies will help the lab understand the mechanisms by which different PMCAs are targeted and retained in specific membranes and how they contribute to the control of Ca2+. In turn, this will contribute to fighting diseases caused by defects in local calcium regulation, such as hearing loss and neuronal degeneration in aging.
Representative recent publications
- Padanyi R, et al. Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells. The Journal of Biological Chemistry. 2010;285:31704.
- Enyedi A, Strehler EE. Regulation of apical membrane enrichment and retention of plasma membrane Ca2+ ATPase splice variants by the PDZ-domain protein NHERF2. Communicative and Integrative Biology. 2011;4:340.
- Antalffy G, et al. Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells. Biochemical and Biophysical Research Communications. 2011;410:322.