1. Application of N-methyl-D-aspartate receptor nanopore in screening ligand molecules
Shu-Peng Li, Yong-Chao Zhang, Fang-Zhou Hu, Tharani Sabaretnam, Gilles J Guillemin, Ai-Hua Zou Bioelectrochemistry. 2020 Aug;134:107534. doi: 10.1016/j.bioelechem.2020.107534. Epub 2020 Apr 17.
N-methyl-D-aspartate receptors (NMDARs) are crucial for excitatory synaptic transmission in the central nervous system. To study NMDARs more accurately and conveniently, we developed a stable NMDAR nanopore in a planar lipid bilayer. Pharmacological properties were validated using the allosteric modulator Ro 25-6981 and antagonist D-2-amino-5-phosphonopentanoic acid (D-APV). The cyanotoxin β-N-methylamino-L-alanine (BMAA) found in fresh water systems is suspected to be associated with the development of neurodegenerative diseases. Therefore, BMAA and its two isomers L-2, 4-Diaminobutyric acid dihydrochloride (DAB) and N-(2-aminoethyl) glycine (AEG) and an endogenous excitotoxin, quinolinic acid (QA), were studied using the NMDAR nanopores to assess their effects on NMDAR modulation. We demonstrated that the NMDAR nanopore could reliably detect its ligand molecules at the single-channel level. The study also demonstrated the practicability of NMDAR nanopores, and results were validated using two-electrode voltage-clamp (TEVC) recording. Compared with TEVC recording, the NMDAR nanopores conducted ion channel gating at the single-channel level without being affected by other proteins on the cell membrane. The highly sensitive and accurate NMDAR nanopore technique thus has a unique advantage in screening NMDAR ligand molecules that could be associated with neurodegenerative disease.
2. Beta-N-methylamino-L-alanine in the presence of bicarbonate is an agonist at non-N-methyl-D-aspartate-type receptors
C N Allen, P S Spencer, D O Carpenter Neuroscience. 1993 Jun;54(3):567-74. doi: 10.1016/0306-4522(93)90228-8.
Beta-N-Methylamino-L-alanine, a component of the neurotoxic Cycas circinalis plant, activates an ionic current which is antagonized by extracellular Ca2+ but not by the excitatory amino acid receptor antagonists D,L-2-amino-5-phosphonovalerate (10-100 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (1-10 microM). This current was reduced by 50% in 0.5 mM extracellular Ca2+ and 92% in 3.0 mM Ca2+ when compared to those recorded in 0.1 mM Ca2+. Addition of 10 or 20 mM NaHCO3 to beta-N-methylamino-L-alanine (500 microM) potentiated the currents 224% and 578%, respectively. Addition of NaHCO3 to the extracellular Ringers (pH 7.2) shifted the pH to 7.7 (10 mM) or 8.3 (20 mM). beta-N-Methylamino-L-alanine was potentiated by NaHCO3 at pH 7.2, 7.7 and 8.3, but the potentiation with NaHCO3 (20 mM) was larger at pH 8.3 (5.7-fold) compared to pH 7.2 (3-fold). NaHCO3 (20 mM) had no effect on quisqualate-, N-methyl-D-aspartate- or kainate-activated ionic currents. The beta-N-methylamino-L-alanine-NaHCO3-activated currents were reduced 49% by 1 microM and 80% by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione suggesting an agonist action at non-N-methyl-D-aspartate-type receptors. Activity at N-methyl-D-aspartate receptors is unlikely since the beta-N-methylamino-L-alanine-NaHCO3 currents are not antagonized by D,L-2-amino-5-phosphonovalerate (10-100 microM), potentiated by addition of glycine (10 microM) or blocked by extracellular Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)