AMPA Glutamate Receptor Channels

The channel of the glutamate N -methyld -aspartate receptor (NMDAR) transports Ca 2 1 approximately four times more efficiently than that of Ca 2 1 -permeable a -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 1 efflux and Ca 2 1 influx in recombinant NMDAR and Ca 2 1 -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 Ca 2 1 using whole-cell currents and fluorometric techniques (dye overload) with Cs 1 internally and Ca 2 1 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 z 2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca 2 1 and Cs 1 as the reference ions, Ca 2 1 permeability (P Ca /P Cs ) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; P Ca /P Cs < 6.1) and reduced at both higher (110 mM; P Ca /P Cs < 2.6) and lower (0.18 mM; P Ca /P Cs < 2.2) concentrations. P Ca /P Cs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca 2 1 . In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca 2 1 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 Ca 2 1 transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca 2 1 transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca 2 1 , whereas that of AMPAR channels only a single site. key words: Ussing flux-ratio test • Goldman-Hodgkin-Katz current equation • Ca 2 1 permeation • fractional Ca 2 1 currents i n t r o d u c t i o n N -methyld -aspartate receptors (NMDAR) 1 and a -amino3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR) mediate fast excitatory neurotransmission in the mammalian central nervous system. Post-synaptic influx of Ca 2 1 via glutamate receptors (GluR) is thought to be a critical step for the induction of long-term changes in synaptic strength and neurotoxicity (Choi, 1988; Bliss and Collingridge, 1993). Native NMDAR channels are heteromers composed of the constitutive NR1 subunit and one or more of four different NR2 subunits (A, B, C, D; for review, see Hollmann and Heinemann, 1994). All NMDAR subtypes are, with small quantitative differences, highly permeable to Ca 2 1 (Monyer et al., 1994; Burnashev et al., 1995; Schneggenburger, 1996). In contrast, AMPAR subtypes are more diverse in their ability to transport Ca 2 1 (Hollmann et al., 1991; Hume et al., 1991; Burnashev et al., 1992 a ; for review, see Burnashev, 1996). Four different AMPAR subunits have been cloned: GluR-A, -B, -C, and -D (alternatively, GluR1-4) (Hollmann and Heinemann, 1994). Ca 2 1 -impermeable AMPAR channels contain the edited form of the GluR-B subunit, termed GluR-B(R), which contains an arginine (R) at the functionally critical Q/R site. AMPAR channels containing only the GluR-A, -C, or -D subunits, which contain a glutamine (Q) at this position, are Ca 2 1 permeable. In NMDAR channels, the homoloLonnie P. Wollmuth’s present address is Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794-5230. Address correspondence to Dr. Lonnie P. Wollmuth, Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794-5230. Fax: 516-632-6661; E-mail: [email protected] 1 Abbreviations used in this paper: AMPAR, a -amino-3-hydroxy-5methyl-4-isoxazolepropionate receptor; GHK, Goldman-Hodgkin-Katz; GluR, glutamate receptor; HEK 293 cell, human embryonic kidney 293 cell; I-V, current–voltage; NMDAR, N -methyld -aspartate receptor; NMDG, N -methyld -glucamine. on Jauary 3, 2018 jgp.rress.org D ow nladed fom