Abstract
Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.
Highlights
NMDA receptor and Naϩ/Ca2ϩ exchanger are involved in glutamate-induced calcium dysregulation in neurons
In this paper, using live cell fluorescence microscopy applied to rat hippocampal neurons in culture, we show that TATCBD3, in addition to N-methyl-D-aspartate receptor (NMDAR), inhibits NCXrev
TAT-CBD3 Inhibits NMDAR and Attenuates Glutamate-induced Ca2ϩ Dysregulation—In experiments with Fura-2FFloaded rat hippocampal neurons (12–14 days in vitro), compared with vehicle control (Fig. 1A), TAT-CBD3 (10 M) significantly attenuated glutamate-induced Ca2ϩ dysregulation (Fig. 1B), whereas CBD3 without TAT (10 M) that cannot cross the membrane (Fig. 1C) and TAT alone were without effect (Fig. 1D)
Summary
NMDA receptor and Naϩ/Ca2ϩ exchanger are involved in glutamate-induced calcium dysregulation in neurons. Results: CRMP2 interacts with and modulates activity of the NMDA receptor and Naϩ/Ca2ϩ exchanger. Significance: CRMP2 interaction with NMDA receptor and Naϩ/Ca2ϩ exchanger affects their activity and is important for glutamate-induced Ca2ϩ dysregulation. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-D-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca2؉ dysregulation, suggesting the key contribution of CRMP2 in these processes. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. The forward mode of NCX, evaluated in experiments with ionomycin-induced Ca2؉ influx into neurons, was strongly suppressed by TAT-CBD3. CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX.
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