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 Ca²⁺ 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 Ca²⁺ mediated by the plasmalemmal Na⁺/Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.

BACKGROUND AND PURPOSEAn isothiourea derivative (2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB‐R7943), a widely used inhibitor of the reverse Na⁺/Ca²⁺exchanger (NCX_rev), was instrumental in establishing the role of NCX_revin glutamate‐induced Ca²⁺deregulation in neurons. Here, the effects of KB‐R7943 on N‐methyl‐D‐aspartate (NMDA) receptors and mitochondrial complex I were tested.EXPERIMENTAL APPROACHFluorescence microscopy, electrophysiological patch‐clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca²⁺flux measurements were made in isolated rat brain mitochondria.KEY RESULTSKB‐R7943 inhibited NCX_revwith IC₅₀= 5.7 ± 2.1 µM, blocked NMDAR‐mediated ion currents, and inhibited NMDA‐induced increase in cytosolic Ca²⁺with IC₅₀= 13.4 ± 3.6 µM but accelerated calcium deregulation and mitochondrial depolarization in glutamate‐treated neurons. KB‐R7943 depolarized mitochondria in a Ca²⁺‐independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca²⁺in the bath solution. KB‐R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB‐R7943 inhibited 2,4‐dinitrophenol‐stimulated respiration of cultured neurons with IC₅₀= 11.4 ± 2.4 µM. With isolated brain mitochondria, KB‐R7943 inhibited respiration, depolarized organelles and suppressed Ca²⁺uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate.CONCLUSIONS AND IMPLICATIONSKB‐R7943, in addition to NCX_rev, blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB‐R7943 and a better understanding of its neuroprotective action.

Ca²⁺ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca²⁺ uptake machinery. The MCU is a Ca²⁺-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca²⁺ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca²⁺ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca²⁺ ionophore ETH129 significantly accelerated Ca²⁺ uptake. The residual Ca²⁺ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca²⁺ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca²⁺ uptake pathway in brain mitochondria that mediates residual Ca²⁺ influx and induction of PTP in a fraction of the mitochondrial population.