Bleomycin (BLM) is an anti-cancer drug that can induce formation of reactive oxygen species (ROS). To investigate the association between up-regulation of antioxidant enzymes and coenzyme Q(10) (CoQ(10)) in acquired BLM resistance, one BLM-resistant clone, SBLM24 clone, was selected from a human oral cancer cell line, SCC61 clone. The BLM resistance of SBLM24 clone relative to a sub-clone of SCC61b cells was confirmed by analysis of clonogenic ability and cell cycle arrest. CoQ(10) levels and levels of Mn superoxide dismutase, glutathione peroxidase 1, catalase and thioredoxin reductase 1 were augmented in SBLM24 clone although there was also a mild increase in the expression of BLM hydrolase. Suppression of CoQ(10) levels by 4-aminobenzoate sensitized BLM-induced cytotoxicity. The results of suppression on enhanced ROS production by BLM and the cross-resistance to hydrogen peroxide in SBLM24 clone further demonstrated the development of adaptation to oxidative stress during the formation of acquired BLM resistance.
The Ν-methyl-D-aspartate (NMDA) receptor channel is an obligatory heterotetramer formed by two GluN1 and two GluN2 subunits. However, the differential contribution of the two different subunits to channel operation is not clear. We found that the apparent affinity of glycine to GluN1 (K gly ∼ 0.6 μM) is much higher than NMDA or glutamate to GluN2 (K NMDA ∼ 36 μM, K glu ∼ 4.8 μM). The binding rate constant (derived from the linear regression of the apparent macroscopic binding rates) of glycine to GluN1 (∼9.8 × 10(6) M(-1) s(-1)), however, is only slightly faster than NMDA to GluN2 (∼4.1 × 10(6) M(-1) s(-1)). Accordingly, the apparent unbinding rates of glycine from activated GluN1 (time constant ∼2 s) are much slower than NMDA from activated GluN2 (time constant ∼70 ms). Moreover, the decay of NMDA currents upon wash-off of both glycine and NMDA seems to follow the course of NMDA rather than glycine unbinding. But if only glycine is washed off, the current decay is much slower, apparently following the course of glycine unbinding. The apparent binding rate of glycine to the fully deactivated channel, in the absence of NMDA, is roughly the same as that measured with co-application of both ligands, whereas the apparent binding rate of NMDA to the fully deactivated channel in the absence of glycine is markedly slower. In this regard, it is interesting that the seventh residue in the highly conserved SYTANLAAF motif (A7) in GluN1 and GluN2 are so close that they may interact with each other to control the dimension of the external pore mouth. Moreover, specific mutations involving A7 in GluN1 but not in GluN2 result in channels showing markedly enhanced affinity to both glycine and NMDA and readily activated by only NMDA, as if the channel is already partially activated. We conclude that GluN2 is most likely directly responsible for the activation gate of the NMDA channel, whereas GluN1 assumes a role of more global control, especially on the gating conformational changes in GluN2. Structurally, this intersubunit regulatory interaction seems to involve the SYTANLAAF motif, especially the A7 residue.
NMDA receptor channels are characterized by high Ca2+ permeability. It remains unclear whether extracellular Ca2+ could directly modulate channel gating and control Ca2+ influxes. We demonstrate a pore-blocking site external to the activation gate for extracellular Ca2+ and Cd2+, which has the same charge and radius as Ca2+ but is impermeable to the channel. The apparent affinity of Cd2+ or Ca2+ is higher toward the activated (a steady-state mixture of the open and desensitized, probably chiefly the latter) than the closed states. The blocking effect of Cd2+ is well correlated with the number of charges in the DRPEER motif at the external pore mouth, with coupling coefficients close to 1 in double mutant cycle analyses. The effect of Ca2+ and especially Cd2+ could be allosterically affected by T647A mutation located just inside the activation gate. A prominent “hook” also develops after wash-off of Cd2+ or Ca2+, suggesting faster unbinding rates of Cd2+ and Ca2+ with the mutation. We conclude that extracellular Ca2+ or Cd2+ directly binds to the DRPEER motif to modify NMDA channel activation (opening as well as desensitization), which seems to involve essential regional conformational changes centered at the bundle crossing point A652 (GluN1)/A651(GluN2).
N-methyl-D-aspartate (NMDA) receptor channels are activated by glutamate (or NMDA) and glycine. The channels also undergo desensitization, which denotes decreased channel availability, after prolonged exposure to the activating ligands.Glycine apparently has a paradoxical negative effect on desensitization, as the increase in ambient glycine in concentrations required for channel activation would increase sustained NMDA receptor currents. We hypothesized that this classical "glycine-dependent desensitization" could be glycine-dependent activation in essence. By performing electrophysiological recordings and biophysical analyses with
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.