Sobolevsky AI et al. (2007), Subunit-specific contribution of pore-forming d...

XB-ART-36247

J Gen Physiol 2007 Jun 01;1296:509-25. doi: 10.1085/jgp.200609718.

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Subunit-specific contribution of pore-forming domains to NMDA receptor channel structure and gating.

Sobolevsky AI , Prodromou ML , Yelshansky MV , Wollmuth LP .

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N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels that contribute to fundamental physiological processes such as learning and memory and, when dysfunctional, to pathophysiological conditions such as neurodegenerative diseases, stroke, and mental illness. NMDARs are obligate heteromultimers typically composed of NR1 and NR2 subunits with the different subunits underlying the functional versatility of NMDARs. To study the contribution of the different subunits to NMDAR channel structure and gating, we compared the effects of cysteine-reactive agents on cysteines substituted in and around the M1, M3, and M4 segments of the NR1 and NR2C subunits. Based on the voltage dependence of cysteine modification, we find that, both in NR1 and NR2C, M3 appears to be the only transmembrane segment that contributes to the deep (or voltage dependent) portion of the ion channel pore. This contribution, however, is subunit specific with more positions in NR1 than in NR2C facing the central pore. Complimentarily, NR2C makes a greater contribution than NR1 to the shallow (or voltage independent) portion of the pore with more NR2C positions in pre-M1 and M3-S2 linker lining the ion-conducting pathway. Substituted cysteines in the M3 segments in NR1 and NR2C showed strong, albeit different, state-dependent reactivity, suggesting that they play central but structurally distinct roles in gating. A weaker state dependence was observed for the pre-M1 regions in both subunits. Compared to M1 and M3, the M4 segments in both NR1 and NR2C subunits had limited accessibility and the weakest state dependence, suggesting that they are peripheral to the central pore. Finally, we propose that Lurcher mutation-like effects, which were identified in and around all three transmembrane segments, occur for positions located at dynamic protein-protein or protein-lipid interfaces that have state-dependent accessibility to methanethiosulfonate (MTS) reagents and therefore can affect the equilibrium between open and closed states following reactions with MTS reagents.

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Species referenced: Xenopus laevis
Genes referenced: grin1 grin2a grin2c nodal2 tbx2

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	Figure 1. Membrane topology of NMDAR NR1 and NR2C subunits. (A) Topology of a NMDAR subunit. Hydrophobic and presumed Î±-helical membrane spanning segments M1, M3, and M4 and the C-terminal half of the M2 pore loop are shown as gray cylinders. The S1 (N terminal to M1) (highlighted in orange) and S2 (between M3 and M4) (highlighted in magenta) lobes comprise the ligand-binding core, a crystal structure of which exists for NR1-NR2A (Furukawa et al., 2005). The thick black bars indicate regions where cysteines were substituted. In all plots subsequent to Fig. 1, presumed transmembrane segments are highlighted in gray with their extracellular side pointing upward. (B) Sequence alignment of regions encompassing the transmembrane domains in NMDAR NR1 and NR2C subunits. The transmembrane segments M1, M3, and M4 are highlighted in gray, whereas proximal parts of S1 and S2 are highlighted in orange and magenta, respectively. Previously described (M3: W613C-I633C) (Sobolevsky et al., 2002b) and newly made (M1, S526C-F541C; M3-S2 linker, Q634C-V641C; and M4, K790C-V805C) single cysteine substitutions in the NR2C subunit are indicated (CCâ¦CC). Numbering is for the mature protein. For M3, we also used a relative numbering system where the serine (S) in the highly conserved SYTANLAAF motif is designated â0â with positions more N or C terminal indicated by negative or positive numbers, respectively.
	Figure 2. Accessibility of substituted cysteines in NR2C to MTS reagents. (AâC) Protocols to assay accessibility of substituted cysteines in the presence (A and B) or absence (C) of glutamate and glycine using steady-state reactions (see Materials and Methods). (A and B) The examples show whole-cell currents recorded from Xenopus oocytes expressing wild-type (wt) NR1-NR2C (A) or NR1-NR2C(A535C) (B) channels. Currents were elicited by glutamate (200 Î¼M) and glycine (20 Î¼M) (thin lines) at a holding potential (Vh) of â60 mV. MTSEA (A) or MTSET (B) (2 mM, thick lines) was applied for 60 s in the continuous presence of coagonists. (C) NR1-NR2C(A797C) channels were probed with MTSEA (2 mM, thick line) applied for 60 s in the continuous presence of the competitive NMDAR antagonist APV (100 Î¼M, open box). (D) Mean percent change (% change) in glutamate-activated current amplitudes measured before (Ipre) and after (Ipost) exposure to MTSEA (MTSEA + Glu) or MTSET (MTSET + Glu) in the presence of glutamate/glycine, or MTSEA in the absence of coagonists but in the continuous presence of APV (MTSEA â Glu). Left and right pointing bars indicate inhibition and potentiation, respectively (n > 4). For positions with % change = â100, potentiation was stronger than 100%. The MTSEA + Glu data for positions W613C-I633C (W-10 to I+10) in the NR2C M3 are from Sobolevsky et al. (2002b). Filled bars indicate that the value of % change is statistically different from zero. Open-ended box encompassing T640C and V641C indicates that these positions belong to S2.
	Figure 3. Voltage dependence of the modification rates of exposed cysteines in the presence of agonists. (A) Pulsive protocol to assay modification rates of exposed cysteines in the presence of glutamate/glycine. The example shows NR1-NR2C(A527C) channels. Vh was â60 mV. The MTSET application (4 Î¼M, thick line, 1 min) was started 15 s after the beginning and finished 15 s before the end of the glutamate/glycine (thin line) application. The cell was washed for 1 min between agonist applications. Current amplitudes, defining the time course of cysteine modification, were measured during the first 15 s of each glutamate exposure. Single exponential fit of these current amplitudes as a function of cumulative time of MTSET exposure (dashed line) gives the time constant Ï = 78 Â± 2 s. The corresponding rate constant of chemical modification in the presence of agonists, k, was 3205 Â± 82 Mâ1sâ1. (B) Apparent second-order rate constant for chemical modification of NR1-NR2C(A527C) by MTSET in the presence of agonists, expressed in a logarithmic form (â(RT/F)*lnk), as a function of the holding membrane potential, Vh. The k values were estimated using the protocol illustrated in A. The error bars are not shown if smaller than the symbol size. The straight line through the points is a fit with Eq. 2 (see Materials and Methods). The slope of this fit gives zÎ´ = â0.01 Â± 0.02. (C) Mean values of k at Vh = â60 mV and zÎ´ for selected positions in NR2C. Rate constants for substituted cysteine modification were measured for MTSET (squares). SEMs are smaller than the symbol size (n > 4). Positions that belong to the M1 or M4 segments are highlighted in gray.
	Figure 4. Analysis of substituted cysteine reactivity with MTSEA/MTSET in the presence of glutamate/glycine. (A) Helical net diagrams illustrating discrete reactivity of substituted cysteines in and around the M1 (top row), M3 (middle row), or M4 (bottom row) segments of NR1 (left half) and NR2C (right half) subunits with MTS reagents in the presence of agonists (MTSEA/MTSET+Glu). All positions indicated were tested for accessibility. Mutants that did not generate detectable glutamate-activated currents are indicated with an X (NR1(S535C)-NR2C and NR1(Y+1)-NR2C). MTSEA and/or MTSET either inhibited (black circles), potentiated (red circles), or had no effect (no circle) on glutamate-activated currents. The data for NR1 are from Beck et al. (1999) and Watanabe et al. (2002), and that for positions W613C-I633C (F-8 to I+10) in NR2C M3 are from Sobolevsky et al. (2002b). Gray regions denote the hydrophobic segments (M1, M3, and M4) (see Fig. 1 B). We aligned positions in M1 and M3 according to their voltage dependence (Fig. 3) (Sobolevsky et al., 2002b). The dashed line in M3s indicates the approximate boundary between positions with voltage-dependent and -independent reaction rates. Positions in M4 did not show any notable voltage dependence so we aligned them according to sequence homology. Clusters of positions with similar properties are highlighted in light blue (black positions with voltage-independent modification rates), dark blue (black positions with voltage-dependent modification rates), and yellow (red positions). (B) Angular width (see Materials and Methods) of dark and light blue clusters of positions that presumably face the ion conduction pathway.
	Figure 5. Effect of the large-sized MTS reagent PTrEA applied in the presence of glutamate/glycine on alanine-to-cysteine substitutions in SYTANLAAF. (AâC) Example recordings showing the effect of the MTS reagent PTrEA on substituted cysteines in the NR1 (left) or NR2C (right) M3 segments. PTrEA was applied in the presence of glutamate/glycine (thin lines) (see Fig. 2). Homologous positions are aligned horizontally.
	Figure 6. Accessibility of substituted cysteines in the NR1 and NR2C M3 segments to PTrEA applied in the presence of glutamate/glycine. (A) Mean percent change (% change) in glutamate-activated current amplitudes (first row) or in leak currents (Î leak) (second row) measured before and after exposure to PTrEA in the presence of glutamate/glycine. Left and right pointing bars indicate inhibition and potentiation, respectively (n > 4). For positions with % change = â100 or Î leak = â100, potentiation was stronger than 100%. Filled bars indicate that the value of % change or Î leak is statistically different from zero. (B) Helical net analysis of the NR1 and NR2C M3 segments showing discrete accessibility to PTrEA in the presence of glutamate/glycine (PTrEA+Glu). All positions indicated were tested for accessibility to PTrEA. The display is comparable to that in Fig. 4 but expands the definition of red positions to include those that show a significant change in leak current.
	Figure 7. Mg2+ block of leak and glutamate-activated currents. (A and B) Example recordings showing the effect of extracellular Mg2+ (100 Î¼M, thick lines) on leak (initial application) or on glutamate-activated (second application) currents in oocytes injected with wild-type NR1-NR2C (A) or NR1(A+3C)-NR2C (B) mRNA. Thin lines indicate applications of glutamate/glycine. Vh was â60 mV. (B) Mean percent change in leak currents (Î leak) upon application of extracellular Mg2+ (n > 4). A filled bar indicates that the value of Î leak is statistically different from zero.
	Figure 8. Analysis of substituted cysteine reactivity with MTSEA in the absence of glutamate/glycine. (A) Helical net diagrams illustrating discrete reactivity of substituted cysteines in and around the M1 (top row), M3 (middle row), or M4 (bottom row) segments of NR1 (left) and NR2C (right) subunits with MTSEA in the absence of agonists (MTSEA-Glu). The data for NR1 are from Beck et al. (1999), whereas that for NR2C is from Fig. 2 D. Within each helical net the gray region denotes the hydrophobic segment. Only positions reactive in the presence of coagonists (Fig. 4 A) are indicated. The results shown are for steady-state reactions for MTSEA with two exceptions. Positions S452 and T543 in NR1 S1-M1 are accessible in the absence but not in the presence of glutamate (Beck et al., 1999). However, this effect was only marginally statistically significant and it seems highly unlikely that there would be positions accessible in the absence of glutamate (closed state) but not also accessible in the presence of glutamate (closed and open states). We therefore for simplicity did not indicate that these positions were accessible in the absence of glutamate. Positions K790 and N795 do not show reactivity with MTSEA either in the presence or absence of glutamate (Fig. 2 D) but do show a robust reactivity with MTSET both in the presence and absence of glutamate (Fig. 2 D and Fig. 9 B, respectively). The lack of an effect with MTSEA presumably reflects a silent reaction, an action we do not explore further here. (B) Angular width of the NR1 and NR2C M3 dark blue and yellow clusters in the presence (+Glu) or absence (âGlu) of glutamate/glycine.
	Figure 9. Modification rates of exposed cysteines in the absence of glutamate. (A) Pulsive protocol to assay modification rates of exposed cysteines in the absence of glutamate. The example shows NR1-NR2C(A527C) channels (as in Fig. 3 A). Vh was â60 mV. 1 min after a 15-s test glutamate application (thin line), APV (100 Î¼M, open box) was applied for 1.5 min. The MTSET application (5 Î¼M, thick line, 1 min) was started 15 s after the beginning and finished 15 s before the end of the APV exposure. After APV, the cell was washed for 1.25 min before the next test glutamate application. Dashed line illustrates a single exponential fit of the current amplitudes as a function of cumulative time of MTSET exposure (Ï = 93 Â± 3 s) that defines the rate constant of chemical modification in the absence of glutamate, kAPV, which was 2151 Â± 70 Mâ1sâ1. (B) Mean values of k (solid symbols), kAPV (open symbols), and k/kAPV (columns) at Vh = â60 mV for NR1 (red) and NR2C (black) subunits (values for k are from Fig. 3 C). Rate constants for substituted cysteine modification were measured for MTSEA (circles), MTSET (squares), or PTrEA (triangles). SEMs are smaller than the symbol size (n > 4). Crossed symbols represent the kAPV values smaller than 1 Mâ1sâ1. Cut bars on the right plot indicate that the corresponding k/kAPV values are larger than shown since in these instances kAPV â¤ 1 Mâ1sâ1. The data for NR1 positions are from Sobolevsky et al. (2002a). For the NR2C M3 segment, the k values are from Sobolevsky et al. (2002b). Positions that belong to the M1, M2, M3, or M4 segments are highlighted in gray.
	Figure 10. Vertical alignment of M1, M3, and M4 segments in NR1 and NR2C subunits. Discrete representation of reactivity of substituted cysteines with MTS reagents in the presence of agonists. MTSEA or MTSET either inhibited (black) or potentiated (red) glutamate-activated currents or did not change their amplitude (white). Some positions are also labeled red because the MTS reagent strongly altered leak current following PTrEA application. The M1, M3, and M4 segments are highlighted in gray, and the proximal parts of S1 and S2 are highlighted in orange and magenta, respectively (see Fig. 1). The dashed line separates the deep and shallow portions of the outer cavity where substituted cysteines sense and do not sense the transmembrane voltage, respectively. The most intracellular-accessible positions in M1 of NR2C as well as M4 in both NR1 and NR2C are placed at this level though we have no means to verify this positioning (since no positions show voltage-dependent reactivity).

References [+] :

Auerbach, Gating reaction mechanisms for NMDA receptor channels. 2005, Pubmed, Xenbase