Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum

Abstract

SUMMARYThe plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events (Strehler et al, 2007), its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre-and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull down approaches confirmed that PMCA2 interacts with the postsynaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its Cterminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre-and post-synaptic signalling complexes.

Calcium Signaling in Dendritic Spines

“…First, ATP-dependent Ca 2þ pumps, such as the plasmalemmal Ca 2þ ATPase (PMCA), move Ca 2þ across the cell membrane from the cytosol into the extracellular space. PMCA isoforms have been found both in the postsynaptic density and localized to spines, placing them appropriately to rapidly clear Ca 2þ from the spine head (Garside et al 2009;Burette et al 2010;Kenyon et al 2010). The high expression of PMCA in pyramidal neurons may explain the rapid Ca 2þ clearance capacity of these cells (Jensen et al 2004;Kip et al 2006).…”

Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

“…Although the physiological consequences of the PSD-95/PMCA4b interaction remain to be determined, PSD-95 has also been reported to induce clustering of other proteins (such as potassium channels and neurotransmitter receptors) that play an important role in the regulation of synaptic signal transduction [54][55][56] , which suggests that PMCA4b participates in the regulation of calcium signaling in the synaptic nerve terminals via PSD-95-induced clustering. In support of the function of PMCA as a regulator of calcium signaling at synapses, Garside et al [57] have analyzed PMCA interactions in synapse-enriched brain tissue from rats, and have found that PMCA2 interacts with the postsynaptic protein PSD-95, and the NMDA glutamate receptor subunits NR1 and NR2a. At the pre-synapse, PMCA2 interacts with the presynaptic protein syntaxin-1A.…”

Contribution of plasma membrane Ca2+ATPase to cerebellar synapse function

“…Subsequent biochemical fractionation approaches have revealed that whilst not enriched, PMCA2 is present in the pre-synapse membrane web fraction, prepared from cerebellar cortex. Furthermore, PMCA2 makes molecular interactions within synapse-enriched protein complexes that contain syntaxin 1A, one of the components of the pre-synaptic, calciumdependent release machinery [8] . The molecular and functional Ca 2+ imaging evidence [9] also points to an important contribution from PMCA2 to pre-synaptic PF function in the cerebellum (see below).…”

“…Further studies will be required to determine this. However, the longer C-terminus of the PMCA2b isoform seems to be functionally important in the cerebellum as its PDZ-binding sequence is capable of interacting with the synaptic PDZdomain-containing protein, PSD95 (and also PSD97), but not SAP102 [8] . This interaction, and the promiscuity of the PMCA2b PDZ sequence, was originally identified in the forebrain [12] and provided the first evidence that PMCA2b could play an important signalling role at synapses.…”

“…This interaction, and the promiscuity of the PMCA2b PDZ sequence, was originally identified in the forebrain [12] and provided the first evidence that PMCA2b could play an important signalling role at synapses. Subsequently, we have identified subunits (NR1 and NR2a) of the NMDA receptor subtype of glutamate receptor as important components of the complex of PMCA2 and PSD95 both in cerebellum and forebrain [8] .…”