Structure-function analysis of PMCA-Neuroplastin/Basigin complexes, the native Ca2+ pump(s) of the plasma membrane

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Calcium (Ca2+) signaling controls a broad variety of cellular processes and reaction pathways ranging from transmitter release and contraction to regulation of enzymatic activities and/or cell motility: These processes are switched on by influx of Ca2+ into the cytosol, and they are switched off by the concerted action of various Ca2+ transporters, most importantly Ca2+-ATPases (or pumps) that are located in the sarco/endoplasmic reticulum (SERCA) or the plasma membrane (PMCA). We have recently identified PMCAs in any type of cell as bimolecular complexes assembled from two ATPase subunits (PMCAs1-4) and two single-span membrane proteins neuroplastin or basigin. Co-assembly of the latter with the PMCA proteins is obligatory for both stabilization and trafficking of the complexes, as well as for their substrate transport. In fact, binding of neuroplastin or basigin increases the rates of Ca2+-transport by more than two orders of magnitude, thus promoting Ca2+-clearing within 10s of milliseconds rather than seconds, as previously assumed. The structural and functional basis of this profoundly increased transport activity, as well as the role of the two auxiliary subunits for the cell-physiology of native PMCAs are currently unknown. This proposal aims at a comprehensive understanding of the structural and functional basis for the formation and operation of PMCA-neuroplastin/basigin complexes. For this purpose we will pursue the following closely interrelated aims and experimental approaches/steps: (1) Elucidate 3D-structures of PMCA2-neuroplastin and PMCA4-basigin complexes, and of PMCA2 in auxiliary subunit-free ‘apo’ state by cryo-EM, (2) Perform structure-function analyses of the Ca2+-transport in PMCA-neuroplastin/basigin complexes, (3) Unravel the interactome of native PMCAs with emphasis on interactions via the Ig-domains of neuroplastin and basigin.