Identification of a High Affinity Divalent Cation Binding Site near the Entrance of the NMDA Receptor Channel (original) (raw)

Analyses of whole-cell current-voltage profiles show Department of Biophysical Sciences that substitution of a Q at this site results in receptors State University of New York at Buffalo with a higher affinity for Ca 2ϩ and a lower affinity for Mg 2ϩ Buffalo, New York 14214 (Burnashev et al., 1992b; Mori et al., 1992). Changes in Ca 2ϩ and Mg 2ϩ block of single channel currents from recombinant NR1-NR2A receptors with N-to-Q muta-Summary tions at the QRN site have also been noted (Ruppersberg et al., 1993). In cortical neurons, Mg 2ϩ binds to a site in Single channel currents from recombinant N-methylthe channel that is about one third through the electric D-aspartate (NMDA) receptors having an N-to-Q mutafield of the membrane from the intracellular side (Johntion in M2 reveal a divalent cation binding site that is son and Ascher, 1990). By examining the ability of differnear the entrance of the pore ‫2.0ف(‬ through the electric ent-sized cations to permeate the pore, it has been estifield). Ca 2؉ rapidly binds to this site and readily permemated that the cross-sectional area of the narrowest ates the channel, while Mg 2؉ binds more slowly and region of the pore is ‫32ف‬ Å 2 (Zarei and Dani, 1995; Vildoes not permeate as readily. In wild-type receptors, laroel et al., 1995). Mg 2؉ also blocks the current by occupying a site that The approach we have taken to studying Ca 2ϩ selecis ‫6.0ف‬ through the field. When the more external site tivity in NMDA receptors is to determine the rate conis occupied by Ca 2؉ , the conductance of the pore to stants of Ca 2ϩ and Mg 2ϩ association to, dissociation Na ؉ is reduced but not abolished, perhaps by an elecfrom, and permeation through mutant and wild-type trostatic blocking mechanism. The site serves to enchannels. To estimate these values, we analyze singlerich the fraction of NMDA receptor current carried channel current amplitudes as a function of the extracelby Ca 2؉. lular concentration of Ca 2ϩ or Mg 2ϩ. In particular, mean amplitudes, open channel noise spectra, and current-Introduction voltage profiles each provide useful information on the kinetics and position of Ca 2ϩ (or Mg 2ϩ) occupancy within Interactions of Ca 2ϩ and Mg 2ϩ with the N-methyl-Dthe pore. The results indicate that mutant receptors bind aspartate (NMDA) receptor channel are important deter-Ca 2ϩ and Mg 2ϩ with a very high affinity, and that main minants of the physiological function of these synaptic and substates of the mutant pore are differentially sensireceptors. Under physiological conditions, Ca 2ϩ constitive to both ions. We conclude that in addition to the tutes ‫%01ف‬ of the total current (Mayer and Westbrook, well-known site of Mg 2ϩ blockade deep within the pore, 1987; Jahr and Stevens, 1993; Schneggenberger et al., NMDA receptors have a divalent cation binding site that 1993; Burnashev et al., 1995). The Ca 2ϩ that enters the is near the external mouth of the pore. When Ca 2ϩ is cell through NMDA receptors acts as a second messenbound to this site, the Na ϩ conductance is reduced but ger that can trigger long term potentiation (Bliss and not abolished, perhaps by an electrostatic mechanism. Collingridge, 1993), cell-death (Choi and Rothman, The external binding site is an important feature of Ca 2ϩ-1990), and synaptic plasticity (Constantin-Paton, 1990; selectivity in NMDA receptors, because it determines reviewed by McBain and Mayer, 1994). Mg 2ϩ blocks the fraction of the NMDA receptor current that is carried open NMDA receptors, and the relief of this block by by Ca 2ϩ. an appropriately-timed depolarization may allow receptors to act as 'coincidence-detectors' (Nowak et al., 1984; Ascher and Nowak, 1988; Johnson and Ascher, Results 1990). To understand further the molecular events that allow Ca 2ϩ permeation and Mg 2ϩ block of the NMDA Nomenclature receptor pore, we have examined the conductance Recombinant NMDAR were expressed in oocytes via properties of single-channel currents from recombinant injection of mouse 1 and ⑀2 subunit cRNAs, which correceptors. respond to NR1-NR2B subunits of the rat. In the wild Although the details of the molecular structures that type, there is an N at position 598 of 1 and at position mediate Ca 2ϩ and Mg 2ϩ binding, permeation, and block 589 of ⑀2. Because both subunits have an N at the QRN are not known, some salient features of the NMDA pore site, we will refer to NMDA receptors constituted from have been determined. Glutamine (Q), arginine (R), and wild-type subunits as N-N receptors. Receptors made asparagine (N) residues at an homologous position (the from subunits in which the N residues at the QRN sites QRN site) in the M2 segment of NMDA (Burnashev et al., of both types of subunit were replaced by a Q will be 1992b; Mori et al., 1992) and other ionotropic glutamate called Q-Q receptors. receptor subunits (Jonas and Burnashev, 1995; Sakurada et al., 1993; Burnashev et al., 1992a; Verdoorn et al., Properties of N-N and Q-Q Receptors 1991; Hume et al., 1991) have been shown to influence in the Absence of Divalent Cations divalent cation selectivity. Wild-type NMDA receptors First, we determined the conductance properties of N-N subunits have an N at the QRN site. Recombinant recepand Q-Q receptors in pure Na ϩ solutions. Figure 1A tors resemble native hippocampal NMDA receptors with shows single-channel currents recorded from N-N reregard to Ca 2ϩ permeability and Mg 2ϩ block (Jahr and ceptors in the absence of divalent cations. At a mem