Different mechanisms of Ca2+ transport in NMDA and Ca2+-permeable AMPA glutamate receptor channels.
The channel of the glutamate N-methyl-D-aspartate receptor (NMDAR) transports Ca2+ approximately four times more efficiently than that of Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs+ efflux and Ca2+ influx in recombinant NMDAR and Ca2+-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca2+ using whole-cell currents and fluorometric techniques (dye overload) with Cs+ internally and Ca2+ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was approximately 2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca2+ and Cs+ as the reference ions, Ca2+ permeability (PCa/PCs) was concentration dependent, being highest around physiological concentrations (1-1.8 mM; PCa/PCs approximately 6.1) and reduced at both higher (110 mM; PCa/PCs approximately 2.6) and lower (0.18 mM; PCa/PCs approximately 2.2) concentrations. PCa/PCs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3-110 mM Ca2+. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca2+ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca2+ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca2+ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca2+, whereas that of AMPAR channels only a single site.
Wollmuth
L P
LP
Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, D-69120 Heidelberg, Germany. lwollmuth@brain.neurobio.sunysb.edu
Sakmann
B
B
D002118:Calcium / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004594:Electrophysiology; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D018091:Receptors, AMPA / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*
D002712:Chlorides; D005971:Glutamates; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D002586:Cesium; C014574:gamma-glutamylmethylamide; D018698:Glutamic Acid; C028019:cesium chloride; D002118:Calcium