Lonnie P. Wollmuth

Structure of the NMDA Receptor Channel M2 Segment Inferred from the Accessibility of Substituted Cysteines

The structure of the NMDA receptor channel M2 segment was investigated by probing the extracellular and cytoplasmic faces of cysteine-substituted NR1-NR2C channels with charged sulfhydryl-specific reagents. The pattern of accessible positions suggests that the M2 segment forms a channel-lining loop originating and ending on the cytoplasmic side of the channel, with the ascending limb in an alpha-helical structure and the descending limb in an extended structure. A functionally critical asparagine (N-site) is positioned at the tip of the loop, and a cluster of hydrophilic residues of the descending limb, adjacent to the tip, forms the narrow constriction of the channel. An apparent asymmetric positioning of the NR1- and NR2-subunit N-site asparagines may account for their unequal role in Ca2+ permeability and Mg2+ block.

Angiotensin II inhibits calcium and M current channels in rat sympathetic neurons via G proteins

We characterized inhibition of N-type Ca2+ and M current K+ channels in rat superior cervical ganglion neurons by angiotensin II (angioII) using the patch clamp. Of 120 neurons, 97 showed inhibition of ICa (mean 32%), which was slow in onset and very slow to reverse under whole-cell recording conditions. This inhibition was blocked by the AT1 receptor antagonist losartan, attenuated by inclusion of 2 mM GDP-beta-S in the pipette, mostly pertussis toxin insensitive, half-sensitive to N-ethylmaleimide, and wholly voltage independent. With 20 mM instead of 0.1 mM BAPTA in the pipette, the inhibition was strongly attenuated; however, we detected no angioII-induced [Ca2+]i signal using the fluorescent indicator indo-1. IBa from cell-attached patches was reduced by bath-applied angioII (mean 33%), suggesting use of a diffusible cytoplasmic messenger. M currents were inhibited by angioII in 8 of 11 neurons (mean 50%) cultured overnight. Hence, a second agonist, angioII, may share the slow, second messenger-utilizing, pertussis toxin-insensitive signaling pathway used by muscarinic agonists.

Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+

1 The voltage‐dependent block of N‐methyl‐D‐aspartate (NMDA) receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set of different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild‐type and mutant NR1‐ and NR2A‐subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2 In wild‐type channels, the voltage dependence (δ) of the block by Mg2+ was concentration dependent with values of δ of ≈0.58 in 0.01 mM and ≈0.82 in 0.07 mM and higher concentrations. Under biionic conditions with high extracellular Mg2+ and K+ as the reference ion, Mg2+ weakly permeated the channel. Over intermediate potentials (≈‐60 to ‐10 mV), this weak permeability had no apparent effect on the block but at potentials negative to ≈‐60 mV, it attenuated the extent and voltage dependence of the block. 3 Substitutions of glycine, serine, glutamine or aspartate for the N‐site asparagine in the NR1‐subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg2+ permeability. 4 In channels containing substitutions of glycine, serine or glutamine for the N‐site asparagine in the NR2A‐subunit, the block by Mg2+ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to ≈0.57 in 0.7 mM Mg2+. 5 Equivalent substitutions for the N + 1 site asparagine in the NR2A‐subunit strongly attenuated the block over the entire voltage range. In 0.7 mM Mg2+, the voltage dependence of the block was reduced to 0.50 (glycine), 0.53 (serine) and 0.46 (glutamine). 6 Channels containing substitutions of the N‐site or N + 1 site asparagines in the NR2A‐subunit showed an increased Mg2+ permeability suggesting that these adjacent asparagines form a barrier for inward Mg2+ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues. 7 The adjacent NR2A‐subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg2+ blocks mutant channels. 8 It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A‐subunit asparagines, the N‐site and N + 1 site, but not the N‐site asparagine of the NR1‐subunit, form a critical blocking site for extracellular Mg2+. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N‐site asparagine. The block may involve binding of Mg2+ to these residues.

NMDAR Channel Segments Forming the Extracellular Vestibule Inferred from the Accessibility of Substituted Cysteines

In NMDA receptor channels, the M2 loop forms the narrow constriction and the cytoplasmic vestibule. The identity of an extracellular vestibule leading toward the constriction remained unresolved. Using the substituted cysteine accessibility method (SCAM), we identified channel-lining residues of the NR1 subunit in the region preceding M1 (preM1), the C-terminal part of M3 (M3C), and the N-terminal part of M4 (M4N). These residues are located on the extracellular side of the constriction and, with one exception, are exposed to the pore independently of channel activation, suggesting that the gate is at the constriction or further cytoplasmic to it. Permeation of Ca2+ ions was decreased by mutations in M3C and M4N, but not by mutations in preM1, suggesting a functionally distinct contribution of the segments to the extracellular vestibule of the NMDA receptor channel.

Differential contribution of the NR1- and NR2A-subunits to the selectivity filter of recombinant NMDA receptor channels.

1. The molecular determinants for the narrow constriction of recombinant N‐methyl‐D‐aspartate (NMDA) receptor channels composed of wild‐type and mutant NR1‐ and NR2A‐subunits were studied in Xenopus oocytes. 2. The relative permeability of differently sized organic cations was used as an indicator of the size of the narrow constriction. From measured reversal potentials under bi‐ionic conditions with K+ as the reference solution, permeability ratios were calculated with the Lewis equation. 3. For wild‐type NMDA receptor channels, five organic cations showed clear reversal potentials, with permeability ratios (PX/PK): ammonium, 1.28; methylammonium, 0.48; dimethylammonium (DMA), 0.20; diethylammonium, 0.07; and dimethylethanol‐ammonium, 0.02. 4. Mutation of the N‐site asparagine (N) to glutamine (Q) at homologous positions in either NR1 (position 598) or NR2A (position 595) increased the permeability of DMA relative to wild‐type channels about equally. However, for larger sized organic cations, the NR1(N598Q) mutation had stronger effects on increasing their permeability whereas the NR2A(N595Q) mutation was without effect. These changes in organic cation permeability suggest that the NR1(N598Q) mutation increases the pore size while the NR2A(N595Q) mutation does not. 5. Channels in which the NR1 N‐site asparagine was replaced by the smaller glycine (G), NR1(N598G)‐NR2A, showed the largest increase in pore size of all sites examined in either subunit. In contrast, in the NR2A‐subunit the same N‐site substitution to glycine produced only small effects on pore size. 6. For the NR2A‐subunit, an asparagine residue (position 596) on the C‐terminal side of the N‐site, when mutated to larger or smaller sized amino acids, produced large, volume‐specific effects on pore size. The mutant channel NR1‐NR2A(N596G) had the largest increase in pore size of all sites examined in the NR2A‐subunit. In contrast, mutation of the homologous position in the NR1‐subunit had no effect on pore size. 7. The cross‐sectional diameter of the narrow constriction in wild‐type NMDA receptor channels was estimated to be 0.55 nm. The pore sizes of the NR1(N598G)‐NR2A and NR1‐NR2A(N596G) mutant channels increased to approximately 0.75 and 0.67 nm, respectively. The double mutation, NR1(N598G)‐NR2A(596G), increased the pore size to approximately 0.87 nm, essentially the sum of the increase produced by the individual mutations. 8. It is concluded that both the NR1‐ and NR2A‐subunits contribute to the narrow constriction of NMDA receptor channels with asparagines located at non‐homologous positions. The major determinants of the narrow constriction in NMDA receptor channels are the NR1 N‐site asparagine and an asparagine adjacent to the NR2A N‐site.

Facilitation of currents through rat Ca2+‐permeable AMPA receptor channels by activity‐dependent relief from polyamine block

1 In outside‐out patches excised from human embryonic kidney (HEK) 293 cells expressing Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate receptor (AMPAR) channels, currents activated by 1 ms glutamate pulses at negative membrane potentials facilitated during and following a repetitive (2 to 100 Hz) agonist application. The degree of facilitation depended on subunit type, membrane potential and stimulation frequency being antagonized by a slow recovery from desensitization. 2 Activity‐dependent current facilitation occurred in Ca2+‐permeable but not in Ca2+‐impermeable AMPAR channels. Current facilitation, however, does not depend on Ca2+ flux. Rather it reflects a relief from the block of Ca2+‐permeable AMPARs by intracellular polyamines since facilitation occurred only in the presence of polyamines and since facilitated currents had a nearly linear current‐voltage relation (I‐V). 3 Relief from polyamine block was use dependent and occurred mainly in open channels. The relief mechanism was determined primarily by membrane potential rather than by current flow. 4 In closed channels the degree of polyamine block was independent of membrane potential. The voltage dependence of the rate of relief from the block in open channels rather than the voltage dependence of the block underlies the inwardly rectifying shape of the I‐V at negative potentials. 5 Currents through native Ca2+‐permeable AMPAR channels in outside‐out or nucleated patches from either hippocampal basket cells or a subtype of neocortical layer II nonpyramidal cells also showed facilitation. 6 It is concluded that a use‐dependent relief from polyamine block during consecutive AMPAR channel openings underlies current facilitation. This polyamine‐AMPAR interaction may represent a new activity‐dependent postsynaptic mechanism for control of synaptic signalling.

Intracellular Mg2+ interacts with structural determinants of the narrow constriction contributed by the NR1-subunit in the NMDA receptor channel

1 N‐methyl‐D‐aspartate (NMDA) receptor channels are blocked by intracellular Mg2+ in a voltage‐dependent manner. Amino acid residues positioned at or near the narrow constriction that interact with intracellular Mg2+ were identified in recombinant NR1‐NR2A channels expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2 In the absence of extracellular Ca2+, the block of wild‐type channels by intracellular Mg2+ measured using macroscopic currents showed a voltage dependence (δ) of around 0.38 and a voltage‐independent affinity for the channel of 4 mM. These parameters were independent of the Mg2+ concentration (0.05‐10 mM), and were indistinguishable from those found for the reduction of single channel amplitudes under the same ionic conditions. Under biionic conditions with high intracellular Mg2+ and K+ extracellularly, Mg2+ was weakly permeant. Mg2+ efflux, however, attenuated the block only at positive potentials (> +80 mV). 3 Substitutions of the N‐site asparagine in the NR1‐subunit altered intracellular Mg2+ block over physiological membrane potentials (+10 to +50 mV). Substitution of glycine, glutamine or serine attenuated the extent of block whereas the negatively charged aspartate enhanced it, consistent with the side chain of the native asparagine at this position contributing to a blocking site for intracellular Mg2+. 4 Substitutions of the N‐site or N + 1 site asparagine in the NR2A‐subunit, which form a blocking site for extracellular Mg2+, also altered the block by intracellular Mg2+. However, for the NR2A‐subunit N‐site asparagine, the block was reduced but only at non‐physiological high potentials (> +70 mV). 5 The NR2A‐subunit N + 1 site asparagine, which together with the NR1‐subunit N‐site asparagine forms the narrow constriction of the channel, also contributed to a blocking site for intracellular Mg2+. However, it did so to a lesser extent than the NR1‐subunit N‐site and in a manner different from its contribution to a blocking site for extracellular Mg2+. 6 It is concluded that intracellular Mg2+ interacts with residues that form the narrow constriction in the NMDA receptor channel with the N‐site asparagine of the NR1‐subunit representing the dominant blocking site. Thus, intracellular Mg2+ interacts with different asparagine residues at the narrow constriction than extracellular Mg2+, although the two blocking sites are positioned very close to each other.

The ion-conducting pore of glutamate receptor channels

Ionotropic glutamate receptors (GluRs) mediate the postsynaptic response at most excitatory synapses in the brain. A variety of G1uR channel subtypes provides these synapses with a repertoire of distinct computational properties, many of which arise from ionic interactions with the channel pore. In both N-methyl-D-aspartate receptor (NMDAR) and a-amino-3-hydroxy-5methylisoxazole-4-propionic acid receptor (AMPAR) channels, a voltage-dependent blocking process renders the response of the channel dependent on the concurrent activity of the synapse. The voltage dependence of extra-cellular Mg2+ block in NMDAR (Mayer et al. 1984; Nowak et al. 1984) and cytoplasmic polyamine block in AMPAR channels (Bowie and Mayer 1995; Donevan and Rogawskj 1995; Isa et al. 1995; Kamboj et al. 1995; Koh et al.1995) allows these transmembrane proteins to integrate pre-and postsynaptic signals. Furthermore, Ca2+ influx through G1uR channels can trigger long-lasting changes in synaptic strength (Bliss and Collingridge 1993) but can also lead to cell death under pathological conditions (Choi 1988; Meldrum and Garthwaite 1990). The amount of Ca2+ entering a neuron during receptor activation may determine the nature and persistence of the postsynaptic response (Berridge 1998). Thus, the control of Ca2+ influx by voltage-dependent blocking mechanisms is essential for the role of GluRs in synaptic plasticity as a basis of higher brain functions, such as learning and memory. Given the physiological and pathophysiological relevance of ionic mechanisms in G1uR function, understanding the structural basis of ion permeation and blocking in these receptor channels is of the utmost interest.