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Title: Intracellular modulation of single NMDA receptor channels in the rat hippocampus
Author: Rycroft, Beth Kathryne
ISNI:       0000 0001 3543 6291
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2001
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The NMDA receptor is subject to a range of intrinsic modulatory systems. By investigating these pathways it is possible to accumulate knowledge concerning potential therapeutic targets. The NMDA receptor plays a key role in many aspects of brain physiology and pathology, therefore information concerning the control of NMDA receptor activity could prove very useful. Certain second messenger systems including protein kinase C, calcineurin and calmodulin in addition to calcium and the cytoskeletal protein alpha-actinin-2, have been shown to alter NMDA receptor activity. The aim of this study was to investigate the molecular mechanisms of this modulation by means of single channel analysis of native NMDA receptors in hippocampal slices. Outside-out patches were taken from the dentate gyrus cell layer of twelve day-old rats. Steady-state channel activations produced by NMDA (0.1 - 10 μM) and glycine (10 μM) were studied over a range of holding potentials (-80 to -30 mV). Exposure of the intracellular surface of outside-out patches to the PKC and calcineurin inhibitors, calphostin-C (n = 4) and cyclosporin-A (n = 5), did not affect single NMDAR channel characteristics. However application of 1.66 nM active calcineurin (n = 7) to outside-out patches caused a 14% reduction in NMDA receptor mean open time at -60 mV. As calmodulin, a cofactor for calcineurin, was present in these experiments, the ability of calmodulin to change NMDA receptor behaviour was also tested. With the same concentration of calmodulin (12 nM) in the absence of calcineurin, mean open time was reduced by 50% at -60 mV. Therefore inhibition of the NMDA receptor by calmodulin and possibly calcineurin was mediated by a reduction in channel open time. This was concluded to be through an allosteric mechanism rather than channel block, as receptor shut times remained unaffected. The duration of groups of channel openings designated bursts, clusters and superclusters were also reduced in the presence of 12 nM calmodulin (n = 8). The mean total open time and charge passed by bursts, clusters and superclusters were also reduced but the average open probability within these events was unchanged. The observed effects on superclusters suggest that calmodulin inhibits the macroscopic NMDA-mediated current and hence has implications for the synaptic current and integration of synaptic activity. To distinguish between the actions of calmodulin at its two binding sites on the NMDA receptor NR1 subunit (C0 and C1 regions), two concentrations of calmodulin were investigated. At 12 nM active calmodulin, the high affinity binding site, the C1 region, is expected to be predominantly occupied, whereas at 800 nM active calmodulin it is expected that both sites would be saturated. At the high calmodulin concentration, a reduction in single NMDA channel mean open time was less obvious than with the low concentration, 29% as compared to 50%. This was fully reversed by antagonising calmodulin binding at its low affinity C0 site, using the C0 binding cytoskeletal protein alpha-actinin. In addition to verifying the competition between calmodulin and alpha-actinin for the C0 region, these results may imply that the two calmodulin binding sites exhibit negative co-operativity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pharmacology & pharmacy & pharmaceutical chemistry