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Physiological Roles of Calpain 1 Associated to Multiprotein NMDA Receptor Complex.

Averna M, Pellegrini M, Cervetto C, Pedrazzi M, Bavestrello M, De Tullio R, Salamino F, Pontremoli S, Melloni E - PLoS ONE (2015)

Bottom Line: Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR.We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel.Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy; Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy.

ABSTRACT
We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

No MeSH data available.


Related in: MedlinePlus

Effect of calpain inhibition on intracellular localization of NMDAR.(A and B) SKNBE cells untreated (Control, solid line) or treated with 1 μM CI-2 (+CI-2, dashed line) for 24 hours were fixed and NR2B localization was determined by confocal microscopy (see Methods). NR2B signal (green fluorescence) was continuously monitored during cell scanning by using Laser Pix software, as previously reported [54]. Each scanning trail is representative of 20 cells analyzed. (C) Aliquots (100 μL) of cell membranes were collected from the two fractions layered at 10% and 30% sucrose interface (see Methods) and assayed for 5′-nucleotidase activity, in order to determine the fraction containing the plasma membranes (PM) and the one containing internal membranes (IM). The same samples were analyzed by immunoblotting to detect the indicated proteins. (D) The protein bands detected in (C) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments.
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pone.0139750.g004: Effect of calpain inhibition on intracellular localization of NMDAR.(A and B) SKNBE cells untreated (Control, solid line) or treated with 1 μM CI-2 (+CI-2, dashed line) for 24 hours were fixed and NR2B localization was determined by confocal microscopy (see Methods). NR2B signal (green fluorescence) was continuously monitored during cell scanning by using Laser Pix software, as previously reported [54]. Each scanning trail is representative of 20 cells analyzed. (C) Aliquots (100 μL) of cell membranes were collected from the two fractions layered at 10% and 30% sucrose interface (see Methods) and assayed for 5′-nucleotidase activity, in order to determine the fraction containing the plasma membranes (PM) and the one containing internal membranes (IM). The same samples were analyzed by immunoblotting to detect the indicated proteins. (D) The protein bands detected in (C) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments.

Mentions: Inspection by confocal microscopy (Fig 4A) revealed that in resting cells 80–90% of NR2B subunits are localized at the plasma membranes and only 10–20% in the cytoplasm. When the cells were exposed to CI-2, the NR2B subunits were detected almost entirely at the plasma membranes and their level was 2−3-fold increased, as confirmed also by western blot analysis (see Fig 3). Concomitantly, the cytoplasmic fluorescence disappeared almost completely. These findings are suggesting that the accumulation of the native and the 60 kD NR2B species take place at different cell localization. To verify this hypothesis plasma membranes were separated from internal membranes by a sucrose gradient. As shown in Fig 4C and 4D, NMDAR containing NR1 and native NR2B subunits was detectable at the plasma membranes (PM) fraction, whereas NMDAR containing NR1 and digested NR2B subunits was present only in the internal membranes (IM) fraction. These data indicate that NMDAR containing the digested NR2B is rapidly removed from the plasma membrane and internalized in endosomes. Accordingly, it can be assumed that in basal conditions, calpain 1 plays an important role in regulating the level of functional NMDAR through the cleavage of NR2B at the C-terminal region thus favoring the formation of vesicles and the internalization of the receptor.


Physiological Roles of Calpain 1 Associated to Multiprotein NMDA Receptor Complex.

Averna M, Pellegrini M, Cervetto C, Pedrazzi M, Bavestrello M, De Tullio R, Salamino F, Pontremoli S, Melloni E - PLoS ONE (2015)

Effect of calpain inhibition on intracellular localization of NMDAR.(A and B) SKNBE cells untreated (Control, solid line) or treated with 1 μM CI-2 (+CI-2, dashed line) for 24 hours were fixed and NR2B localization was determined by confocal microscopy (see Methods). NR2B signal (green fluorescence) was continuously monitored during cell scanning by using Laser Pix software, as previously reported [54]. Each scanning trail is representative of 20 cells analyzed. (C) Aliquots (100 μL) of cell membranes were collected from the two fractions layered at 10% and 30% sucrose interface (see Methods) and assayed for 5′-nucleotidase activity, in order to determine the fraction containing the plasma membranes (PM) and the one containing internal membranes (IM). The same samples were analyzed by immunoblotting to detect the indicated proteins. (D) The protein bands detected in (C) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592069&req=5

pone.0139750.g004: Effect of calpain inhibition on intracellular localization of NMDAR.(A and B) SKNBE cells untreated (Control, solid line) or treated with 1 μM CI-2 (+CI-2, dashed line) for 24 hours were fixed and NR2B localization was determined by confocal microscopy (see Methods). NR2B signal (green fluorescence) was continuously monitored during cell scanning by using Laser Pix software, as previously reported [54]. Each scanning trail is representative of 20 cells analyzed. (C) Aliquots (100 μL) of cell membranes were collected from the two fractions layered at 10% and 30% sucrose interface (see Methods) and assayed for 5′-nucleotidase activity, in order to determine the fraction containing the plasma membranes (PM) and the one containing internal membranes (IM). The same samples were analyzed by immunoblotting to detect the indicated proteins. (D) The protein bands detected in (C) were quantified as described in Methods. Each value represents the arithmetical mean ± SEM of three different experiments.
Mentions: Inspection by confocal microscopy (Fig 4A) revealed that in resting cells 80–90% of NR2B subunits are localized at the plasma membranes and only 10–20% in the cytoplasm. When the cells were exposed to CI-2, the NR2B subunits were detected almost entirely at the plasma membranes and their level was 2−3-fold increased, as confirmed also by western blot analysis (see Fig 3). Concomitantly, the cytoplasmic fluorescence disappeared almost completely. These findings are suggesting that the accumulation of the native and the 60 kD NR2B species take place at different cell localization. To verify this hypothesis plasma membranes were separated from internal membranes by a sucrose gradient. As shown in Fig 4C and 4D, NMDAR containing NR1 and native NR2B subunits was detectable at the plasma membranes (PM) fraction, whereas NMDAR containing NR1 and digested NR2B subunits was present only in the internal membranes (IM) fraction. These data indicate that NMDAR containing the digested NR2B is rapidly removed from the plasma membrane and internalized in endosomes. Accordingly, it can be assumed that in basal conditions, calpain 1 plays an important role in regulating the level of functional NMDAR through the cleavage of NR2B at the C-terminal region thus favoring the formation of vesicles and the internalization of the receptor.

Bottom Line: Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR.We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel.Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy; Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV, 1-16132, Genova, Italy.

ABSTRACT
We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

No MeSH data available.


Related in: MedlinePlus