Kinetically distinguishable AMPA receptors in rat hippocampus are associated with the loss of glutamate-sensitive conformational transitions.
We describe a stopped-flow method to study alpha-amino-7-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-kainate receptor-mediated Na+ ion flux through native membranes. Resealed plasmalemma vesicles and nerve endings from the rat hippocampus were mixed rapidly with a membrane impermeant form of the fluorescence indicator, sodium binding benzofurane oxazole and the changes in fluorescence intensity in response to various [Glu] on the time scale of 0.04 ms-10 s were monitored at a sampling rate of 6.55 kHz. Inhibitors like ouabain (1 mM) and 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (dizocilpine, 50 microM) enhanced Na+ ion translocation under low-[Na+] and physiological conditions, respectively. Dependence of AMPA-kainate receptor kinetics on [Glu] was described in a model of channel activation by faster and slower desensitizing receptors. The model accounted for almost all of the Na+ ion flux activity in the 30 microM-10 mM range of [Glu]. We found that the values of the initial rate constant for Na+ ion influx, JA, and rate constant for desensitization, alpha, for the faster desensitizing receptor were dependent on data sampling rate, whereas the initial rate constant for Na+ ion flux through the slower desensitizing receptor, JB, varied much less with the sampling rate. These phenomena can be described by (1) a fractal model of short-lived AMPA-kainate receptor channel with many closely spaced states (fractal dimension approximately 1.8) and (2) a model of long-lived AMPA-kainate receptor channel with two discrete states.
Szárics
E
E
Department of Neurochemistry, Institute of Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest.
Nyitrai
G
G
Kovács
I
I
Kardos
J
J
D000818:Animals; D016291:Dizocilpine Maleate / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D006624:Hippocampus / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000737:chemistry* / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000737:chemistry* / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013574:Synaptosomes / Q000378:metabolism*
D016291:Dizocilpine Maleate; D018698:Glutamic Acid; D010042:Ouabain; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D012964:Sodium