Na 1 Occupancy and Mg 2 1 Block of the N - methyl - d - aspartate Receptor Channel

The effect of extracellular and intracellular Na 1 on the single-channel kinetics of Mg 2 1 block was studied in recombinant NR1-NR2B NMDA receptor channels. Na 1 prevents Mg 2 1 access to its blocking site by occupying two sites in the external portion of the permeation pathway. The occupancy of these sites by intracellular, but not extracellular, Na 1 is voltage-dependent. In the absence of competing ions, Mg 2 1 binds rapidly ( . 10 8 M 2 1 s 2 1 , with no membrane potential) to a site that is located 0.60 through the electric field from the extracellular surface. Occupancy of one of the external sites by Na 1 may be sufficient to prevent Mg 2 1 dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the Mg 2 1 dissociation rate constant is . 10 times greater than the Mg 2 1 permeation rate constant, and the Mg 2 1 equilibrium dissociation constant is z 12 m M. Physiological concentrations of extracellular Na 1 reduce the Mg 2 1 association rate constant z 40-fold but, because of the “lock-in” effect, reduce the Mg 2 1 equilibrium dissociation constant only z 18-fold. key words: ion binding sites • magnesium • channel blockade • permeation • selectivity I N T R O D U C T I O N At synapses, the channel domain of the N -methyld -aspartate receptor (NMDAR) 1 interacts with several different metal cations, including Na 1 , K 1 , Ca 2 1 , and Mg 2 1 . These interactions have important physiological consequences. Activation of NMDARs depolarizes dendrites, with Na 1 and K 1 carrying the bulk of the current. Ca 2 1 entry into dendrites via NMDARs regulates synaptic strength and plasticity (Maren and Baudry, 1995; Asztely and Gustafsson, 1996). Voltage-dependent Mg 2 1 block of NMDARs may allow these receptors to respond specifically to contemporaneous excitatory inputs and, thus, serve as a substrate for Hebbian learning. Given such critical functions, it is likely that the NMDAR has specific structures that govern the passage of Na 1 , K 1 , Ca 2 1 , and Mg 2 1 through its ion permeation pathway. These four cations interact with the NMDAR protein over time scales that span more than four orders of magnitude. Na 1 and K 1 are highly permeable and pass through the pore rapidly. In symmetric, 100-mM divalent cation-free solutions, the NMDAR conductance is z 70 pS for both of these ions, which means that at 2 60 mV, Na 1 and K 1 interact with the channel for , 0.04 m s. The contact between these ions and the protein is too brief to be resolved as discrete events in the single-channel record. Instead, the interaction of Na 1 and K 1 with the NMDAR pore has been probed mainly using current-voltage relationships (Mayer et al., 1984; Nowak et al., 1984; Cull-Candy and Usowicz, 1987) and by measuring their effects on channel block (Chen and Lipton, 1997; Antonov et al., 1998; Antonov and Johnson, 1999). Under physiological conditions, z 10–15% of the NMDAR current is carried by Ca 2 1 (Burnashev et al., 1995). This divalent cation moves through the wildtype (NR1-NR2B) channel z 100 times more slowly than Na 1 , i.e., with a rate constant of z 10 6 s 2 1 (Premkumar and Auerbach, 1996). Thus, Ca 2 1 resides in the permeation pathway for z 1 m s. Information regarding Ca 2 1 transmission has been derived mainly from measurements of macroscopic currents (Mayer and Westbrook, 1987; Iino et al., 1990; Jahr and Stevens, 1993; Sharma and Stevens, 1996b), single-channel currents (Iino et al., 1997), or fluxes (Burnashev et al., 1995). However, certain mutations reduce the Ca 2 1 permeation rate constant to such an extent that its binding and unbinding are manifest as excess open channel noise (Premkumar and Auerbach, 1996). Mg 2 1 is the slowpoke of the group. This divalent cation typically dwells in the pore for . 100 m s (Ascher and Nowak, 1988; Jahr and Stevens, 1990). Occupancy of the channel by one Mg 2 1 eliminates conduction by other ions and generates a discrete gap in the singlechannel current record. Therefore, the microscopic Address correspondence to Anthony Auerbach, Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, SUNY at Buffalo, 124 Sherman Hall, Buffalo, NY 14214. Fax: (716) 829-2569; E-mail: [email protected] 1 Abbreviations used in this paper: MSC, model selection criterion; NMDAR, N -methyld -aspartate receptor . on Jne 0, 2017 D ow nladed fom Published February 26, 2001