The goal of this research is to elucidate the fundamental mechanisms by which Na+ transport influences Ca2+ homeostasis and signaling, in neurons and glia. Neurons and glia both express Na+ pumps with the a1 isoform of the catalytic (a) subunit and Na+ pumps with either the ot2 (glia) or a3 (neurons) isoform. Na+ pumps with a2 or a3 isoforms are localized to plasma membrane-endoplasmic reticulum (PM-ER) junctional complexes (""""""""PLasmERosomes"""""""") and are coupled to PM Na/Ca exchangers (NCX) and, thus, to cytosolic ([Ca2+]CYr) and ER Ca2+ concentration and Ca2+ signal regulation in these cells. Critical questions are: How are the a2 and oc3 Na+ pumps sorted and tethered to their appropriate PM destinations? And, what are the local and global functional consequences of this special organization? There are four Specific Aims:
Aim 1. To determine how Na+ pump a2 and a3 subunits are targeted and tethered to their appropriate PM locations, a subunit chimeras (e.g., part a2 and part a1, and vice-versa), WT and mutated a truncations, and ankyrin B knockout mice will be used to test the hypothesis that ot2and a3 subunits are targeted to PLasmERosomes by specific N-terminal amino acid (AA) sequences and are tethered by ankyrin B.
Aim 2. To determine whether the sub-PM Ca2+ concentration at PM-ER junctions is controlled independently of """"""""bulk"""""""" [Ca2+]CYT- Novel near-membrane Ca2+ indicators (FFP-18 and G-CaMP-2, an engineered protein targeted to the PM- ER junction) will be used to test, directly, the idea that PM-ER junctional space Ca2+ is regulated by oc2/a3 Na+ pumps and NCX1 in astrocytes and neurons.
Aim 3. To determine how linkage of the Na+ pump a2 subunit isoform, NCX1, and ankyrin B contributes to their central roles in local Ca2+ regulation and global Ca2+ signaling in astrocytes.
Aim 4. To determine the roles of the Na+ pump and NCX in Ca2+ efflux from neuronal dendrites and nerve terminals and how these processes influence neuronal function.
For Aims 3 and 4, null mutant mice, and molecular biological and pharmacological tools will be used to test the hypothesis that structural linkage of key PM Na+ and Ca2+ transporters in PLasmERosomes enables them to serve critical functionally-coupled roles in Ca2+ regulation and signaling in neurons (Aim 4) and in astrocytes (Aim 3). These studies will shed new light on specific mechanisms that regulate normal Ca2+ homeostasis and signaling, and that may go awry during hypoxia/ischemia and other brain pathologies.
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|Song, Hong; Thompson, Scott M; Blaustein, Mordecai P (2013) Nanomolar ouabain augments Ca2+ signalling in rat hippocampal neurones and glia. J Physiol 591:1671-89|
|Blaustein, Mordecai P (2013) Livin' with NCX and lovin' it: a 45 year romance. Adv Exp Med Biol 961:3-15|
|Song, Hong; Lee, Moo Yeol; Kinsey, Stephen P et al. (2006) An N-terminal sequence targets and tethers Na+ pump alpha2 subunits to specialized plasma membrane microdomains. J Biol Chem 281:12929-40|
|Lee, Moo Yeol; Song, Hong; Nakai, Junichi et al. (2006) Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor. Proc Natl Acad Sci U S A 103:13232-7|
|Golovina, Vera A (2005) Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum. J Physiol 564:737-49|
|Lencesova, Lubomira; O'Neill, Andrea; Resneck, Wendy G et al. (2004) Plasma membrane-cytoskeleton-endoplasmic reticulum complexes in neurons and astrocytes. J Biol Chem 279:2885-93|
|Golovina, Vera A; Song, Hong; James, Paul F et al. (2003) Na+ pump alpha 2-subunit expression modulates Ca2+ signaling. Am J Physiol Cell Physiol 284:C475-86|
|Gonzalez-Serratos, Hugo; Chang, Ruzhang; Rozycka, Monika et al. (2003) Role of the T-system and the Na-K pump on fatigue development in phasic skeletal muscle. Adv Exp Med Biol 538:543-55; discussion 555|
|Blaustein, Mordecai P; Golovina, Vera A; Song, Hong et al. (2002) Organization of Ca2+ stores in vascular smooth muscle: functional implications. Novartis Found Symp 246:125-37; discussion 137-41, 221-|
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