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Engineering a high-affinity allosteric binding site for divalent cations in kainate receptors
Andrew J R Plested1, Mark L Mayer
1Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Building 35, Room 3B 1002, 35 Lincoln Drive, Bethesda, MD 20892 3712, USA.
Neuropharmacology
|August 9, 2008
Summary
Kainate receptors, crucial for brain function, are regulated by sodium ions. A specific mutation (GluK2 M739D) unexpectedly shows altered ion sensitivity due to divalent cations like calcium and magnesium.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Kainate receptors are ionotropic glutamate receptors that play key roles in synaptic transmission and plasticity.
- These receptors are allosterically modulated by extracellular sodium ions (Na+), influencing their activity.
- Previous studies indicated that mutations in the neurotransmitter binding domain can alter cation sensitivity.
Purpose of the Study:
- To investigate the cation binding properties of the GluK2 M739D mutant, which showed reduced sensitivity to monovalent cations.
- To explore the role of divalent cations (Ca2+ and Mg2+) in modulating the activity of the GluK2 M739D mutant.
- To elucidate the underlying mechanisms of allosteric regulation in kainate receptors.
Main Methods:
- Site-directed mutagenesis to create the GluK2 M739D mutant.
- Electrophysiological recordings to assess receptor activity in solutions with varying cation compositions.
- Biochemical assays to characterize the binding site properties.
Main Results:
- The GluK2 M739D mutant exhibited insensitivity to Na+ substitution by large monovalent cations (Tris, NMDG).
- In Na+-free solutions, low concentrations of Ca2+ and Mg2+ modulated the mutant receptor's activity.
- EDTA inhibited the mutant in the presence of low divalent cations, suggesting a role for these ions.
- The binding site characteristics of the mutant were consistent with Ca2+ and Mg2+ binding sites.
Conclusions:
- The apparent insensitivity of the GluK2 M739D mutant to monovalent cations is attributed to allosteric modulation by divalent ions.
- Divalent cations (Ca2+, Mg2+) act as positive allosteric modulators at physiological and sub-physiological concentrations.
- This finding highlights the complex interplay of ions in regulating kainate receptor function.
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