Abstract
Previous studies have indicated that inorganic and organic cations can markedly affect parameters of the function of theN-methyl-d-aspartate receptor ionophore complex. As these effects may involve modulation of agonist binding, the purpose of our study was to investigate the stimulatory effect of mono- and divalent cations on binding properties of glutamate/N-methyl-d-aspartate recognition sites on the N-methyl-d-aspartate receptor complex, using [3H]CGP 39653 as the specific ligand for these sites. In well-washed membranes from rat brain, [3H]CGP 39653 binding sites were present at two affinity states when assayed at 10 mM HEPES·KOH buffer. About 75% of these sites were in a low-affinity state (K d = 210 ± 30 nM) although 25% were in a high-affinity state (K d= 6.4 ± 0.4 nM). Addition of mono- or divalent cations to the incubation medium stimulated [3H]CGP 39653 binding, measured at a radioligand concentration of 4 nM. Maximal increases in binding were to ∼230 and 400% of control, in the presence of mono- and divalent cations, respectively. Values of EC50 for stimulation were 5 to 7 mM for monovalent cations and 0.2 to 0.4 mM for divalent cations. At these concentrations, cations increased the Bmax for the high-affinity population of [3H]CGP 39653 sites and decreased the Bmaxfor low-affinity ones. These findings suggest that, like spermidine, inorganic cations stimulate binding by converting [3H]CGP 39653 binding sites from the low- to high-affinity state.
Footnotes
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Send reprint requests to: Dr. Edythe D. London, Chief, NIDA Brain Imaging Center, 5500 Nathan Shock Drive, Baltimore, MD 21224.
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↵1 Current address: Department of Zoology, University of Maryland, College Park, MD 20742-4415.
- Abbreviations:
- NMDA
- N-methyl-d-aspartate
- MK 801
- dizocilpine
- TCP
- 1-[1-(2-thienyl)cyclohexyl]piperidine
- CPP
- (+)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid
- D-AP5
- D-2-amino-5-phosphonopentanoic acid
- trans-(±)-ACPD
- trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid
- SPD
- spermidine
- EC50
- concentration of salts required to produce half-maximal enhancement of binding
- C
- concentration of added cations
- I
- concentration of inhibitor
- KdK+
- dissociation constant for K+
- KdC
- dissociation constant for added cations
- IC50
- concentration of inhibitor that reduces specific binding by 50%
- n
- Hill coefficient
- Bmaxh0
- density of high affinity binding sites in the absence of added salts
- Kd
- dissociation constant for [3H]CGP 39653
- F
- concentration of radioligand
- Bmaxh
- density of high affinity binding sites in the presence of added salts
- Ki
- dissociation constant of the inhibitor
- MK+
- quantity of modulatory sites bound by K+ (introduced in buffer)
- Mc
- quantity of modulatory sites occupied by added cations
- M0
- quantity of allosteric sites occupied by K+ in the control condition (no added salts)
- Kdc
- dissociation constant for added cations
- K0+
- concentration of potassium introduced in the buffer (12 mM)
- C
- concentration of added cations
- v
- valence of the cation
- Bmax
- density of radioligand binding sites
- Bs
- binding in the presence of added salts
- B0
- binding in the absence of added salts
- M
- total quantity of allosteric salts
- Received October 9, 1996.
- Accepted April 16, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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