Correspondence to: Mark L. Mayer, Building 49, Room 5A78, 49 Convent Drive MSC 4495, Bethesda, MD 20892. Fax:(301) 402-4777 E-mail:mlm{at}helix.nih.gov.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pores of glutamate receptors and K+ channels share sequence homology, suggesting a conserved secondary structure. Scanning mutagenesis with substitution of alanine and tryptophan in GluR6 channels was performed based on the structure of KcsA. Our assay used disruption of voltage-dependent polyamine block to test for changes in the packing of pore-forming regions. Alanine scanning from D567 to R603 revealed reduced rectification resulting from channel block in two regions. A periodic pattern from F575 to M589 aligned with the pore helix in KcsA, whereas a cluster of sensitive positions around Q590, a site regulated by RNA editing, mapped to the selectivity filter in KcsA. Tryptophan scanning from D567 to R603 revealed similar patterns, but with a complete disruption of spermine block for 7 out of the 37 positions and a pM dissociation constant for Q590W. Molecular modeling with KcsA coordinates showed that GluR6 pore helix mutants disrupting polyamine block pack against M1 and M2, and are not exposed in the ion channel pore. In the selectivity filter, tryptophan creates an aromatic cage consistent with the pM dissociation constant for Q590W. A scan with glutamate substitution was used to map the cytoplasmic entrance to the pore based on charge neutralization experiments, which established that E594 was uniquely required for high affinity polyamine block. In E594Q mutants, introduction of glutamate at positions S593L600 restored polyamine block at positions corresponding to surface-exposed residues in KcsA. Our results reinforce proposals that the pore region of glutamate receptors contains a helix and pore loop analogous to that found in K+ channels. At the cytoplasmic entrance of the channel, a negatively charged amino acid, located in an extended loop with solvent-exposed side chains, is required for high affinity polyamine block and probably attracts cations via a through space electrostatic mechanism.
Key Words: AMPA receptor, kainate receptor, polyamines, pore helix, ion channel block
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An accumulating body of evidence suggests that the pore region of glutamate receptor ion channels (GluRs)1 has a similar architecture to that found in K+ channels, sodium channels, calcium channels, hyperpolarization-activated channels, and cyclic nucleotidegated channels (-helical segment (the pore helix), which acts both as a dielectric focusing device for cations as well as holding in place a short loop which in K+ channels forms ion binding sites and acts as the selectivity filter (
Definitive evidence for this architecture is available only for one member of the large gene family of K+ channels, the KcsA channel from Streptomyces lividans, for which a structure was solved by X-ray diffraction to 3.2 Å (
Before X-ray crystallographic analysis of KcsA the "M2" segment (see Fig 1) in glutamate receptors was thought to form a hairpin motif (-helical structure lining the ion channel (
|
The results of our experiments are consistent with a remarkably similar secondary structure of the pore regions of KcsA and GluR6. We also show that a negative charge near the cytoplasmic entrance is an essential requirement for high affinity block by polyamines, and that within an extended loop connecting the pore and inner membrane helices, the position of the charge can be moved over a distance exceeding 17 Å without disrupting block.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular Biology
Site-directed mutagenesis by PCR, preparation of RNA for injection in Xenopus oocytes, and preparation of DNA for transfection of HEK293 cells were performed as described previously (
Oocyte and HEK Cell Preparation
Oocytes were surgically removed from Xenopus laevis (Xenopus One) using aseptic techniques after induction of anesthesia by immersion for 15 min in water containing 3 g/liter tricaine, a protocol approved by the NICHD Animal Care and Use Committee. Animals were killed after a maximum of up to six surgeries. Oocytes were defolliculated by incubation of ovarian fragments for 6090 min with 1.5 mg/ml collagenase dissolved in Ca2+-free solution containing (in mM): 83 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.5. The preparation was thoroughly rinsed with solution containing (in mM): 88 NaCl, 2.5 NaHCO3, 1.1 KCl, 0.4 CaCl2, 0.3 Ca(NO3)2, 0.8 MgCl2, 2.5 mM sodium pyruvate, 10 HEPES, pH 7.3, and 5 µg/ml gentamicin; and stored overnight. Dumont stage VVI oocytes were individually selected and injected with 40 pg to 50 ng of cRNA, as required, and stored at 18°C for 23 d before recording. Intervals of up to 10 d between mRNA injection and recording were used only for mutants that were nonfunctional or expressed low amplitude responses after an initial assay at 23 d.
HEK 293 cells (CRL 1573; American Type Culture Collection) were maintained at a confluency of 7080% in MEM with Earle's salts, 2 mM glutamine, and 10% fetal bovine serum. 24 h after plating at low density (2 x 104 cells/ml) onto the center of 35-mm petri dishes, cells were transfected using the calcium phosphate technique; cotransfection with the cDNA for green fluorescent protein (S65T mutation) helped to identify transfected cells during experiments as described previously (
Recording Conditions and Solutions
Two-electrode voltage clamp recording for Xenopus oocytes was performed using 3 M KCl filled agarose cushion microelectrodes of resistance 0.51.2 M (
Outside-out patches were excised from HEK cells using fire-polished, thin-walled borosilicate glass pipets (25 M) coated with dental wax to reduce electrical noise. Experiments were performed in an external solution containing (in mM): 150 NaCl, 1 KCl, 0.7 BaCl2, 0.8 MgCl2, and 5 HEPES, pH 7.3; and the osmolarity was adjusted to 295 mOsm with sucrose. GluR6 responses were evoked using 50 µM domoic acid, a weakly desensitizing agonist, applied via a stepper motor-based fast perfusion system (
Data Analysis
Procedures in the Igor program (Wavemetrics) were used to generate and analyze conductance-voltage (G-V) plots. First, the reversal potential (Vrev) was estimated using a fifth order polynomial fit to I-V plots for either single (oocytes) or the average of five (HEK cells) leak-subtracted responses. G-V plots were generated from the relationship G = I/(V - Vrev) and used to measure the conductance at +80 and -80 mV. A fit of the following Boltzmann function over the range -100 to +20 mV was used to obtain an initial estimate of the voltage dependence of block by polyamines
where Gmax is the conductance at a sufficiently hyperpolarized potential to produce full relief from block by polyamines, Vm is the membrane potential, Vb the potential at which 50% block occurs, and kb is a slope factor describing the voltage dependence of block. To analyze block over a wider range of membrane potentials for mutants with a low affinity for polyamines, it was necessary to correct for rectification that occurs in polyamine-free conditions (
where Gmax, Vm, Vb , and kb have the same meaning as described above, Vp is the half unblock potential for the second Boltzmann function describing the permeation of polyamines on strong depolarization, and kp is the slope factor describing the voltage dependence of relief from block. A voltage-independent dissociation constant for polyamine block, Kd(0), was calculated from the following relationship:
assuming a cytoplasmic concentration of spermine, spermidine, and possibly other polyamines functionally equivalent to the block produced by 20 µM spermine [Spm] alone (
Numerical values in the text and error bars in graphs indicate mean ± SD, unless noted differently. Unpaired t tests were used to test for significant differences where required.
Immunohistochemistry
4 d after injection with 10 ng cRNA, oocytes were fixed overnight by immersion in 100 mM PBS, pH 7.4, containing 4% paraformaldehyde and 15% of a saturated solution of picric acid; uninjected oocytes from the same preparations were processed identically. The oocytes were cryoprotected by overnight immersion in a solution containing 10 mM PBS, 10% glycerol, 0.008% NaN3, and 25 g/100 ml sucrose. 100-µM sections were cut on a freezing microtome, incubated with 2% Triton in PBS, blocked with 10% goat serum in 0.2% Triton for 2 h, and reacted overnight at 4°C with 1 µg/ml rabbit antiGluR6 antibody (
Online Supplemental Material
A PDB file for the model shown in Fig 8 (below) is available online at: http://www.jgp.org/cgi/content/full/117/4/345/DC1.
|
|
|
|
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We began our experiments by performing sequence alignments for GluR6 (
GluR6 Sequence Mapped onto the KcsA Crystal Structure
The amino acid sequence conservation shown in Fig 1 suggests that GluRs and KcsA might have a similar structure in the pore helix and pore loop (
Alanine Scanning Mutagenesis in the Pore Region of GluR6
Wild-type GluR6 responses show such strong biphasic rectification due to permeable block by cytoplasmic polyamines that there is nearly complete attenuation of outward current flow between 10 and 50 mV (
For the 34 Ala mutants with measurable biphasic rectification because of polyamine block, the mean value for kb was 16.4 ± 1.94 mV-1, with values for Vb ranging from -47.9 to 3.9 mV and Kd(0) values from 0.8 to 26 µM (Table 1). The similar values for kb for these mutants indicate changes in affinity for polyamines but not the electrical location of the binding site(s) (
|
Tryptophan Scanning Mutagenesis Reveals Disruption of a Pore Helix
Substitution of tryptophan from D567 to R603 produced both increases and decreases in polyamine block as well as three nonfunctional mutants. Changes in polyamine block were quantified by analysis of G-V plots as described for results of the alanine scan. Although tryptophan mutants showed much larger changes in polyamine block than observed for alanine substitution, these effects also showed strong positional dependence and, for 14 mutants, Kd(0) values for spermine were within twofold of the wild type (Table 1). For two positions, tryptophan mutants showed stronger block than for wild-type GluR6, an effect not observed for any of the alanine mutants. As reported previously, this effect was dramatic for the mutant Q590W for which the affinity for spermine increased 6.6 x 103-fold (
Similar to the results of the alanine scan, a reduction in polyamine block was the more typical effect recorded for 18 positions substituted with tryptophan. As for the Ala scan mutants, these responses fell into two classes. For 11 positions, there were parallel rightward shifts in G-V plots compared with responses for wild-type GluR6, reflecting a decrease in affinity for spermine with little change in the voltage dependence of block (Fig 3). For these mutants, the mean value for kb was 23.5 ± 5.8 mV-1 with values for Vb ranging from -48.2 to 9.8 mV and Kd(0) values of 3.2 to 25.6 µM (Table 1). Responses for a typical example (M589W), with kb = 18.3 ± 0.5 mV-1, Vb = -8.4 ± 1.3 mV, and Kd(0) = 12.2 ± 0.2 µM (n = 3), are shown in Fig 3 A. For 7 other positions, polyamine block was much more strongly disrupted. This prevented an accurate estimation of Kd(0) at the physiological concentrations of spermine present in the cytoplasm of Xenopus oocytes. G-V plots for mutants at these positions were either outward-rectifying with conductance ratios at +80/-80 mV of 327 ± 57% for G584W (n = 4), 195 ± 7% for G592W (n = 4), and 264 ± 7% for E594W (n = 5), or showed very weak voltage dependence with conductance ratios at +80/-80 mV of 96 ± 2% for T576W (n = 4), 75 ± 5% for N579W (n = 3), 88 ± 4% for S580W (n = 4), and 109 ± 2% for G586W (n = 3). Typical examples are shown in Fig 3 B for S580W and G584W. For these and other mutants with low sensitivity to polyamines, we again note that the Kd(0) is likely to be >100 µM. Since our goal was to measure the pattern of changes produced by scanning mutagenesis with tryptophan, rather than individual Kd(0) values for mutants with very low affinity for polyamines, we did not attempt to characterize these mutants further.
For three mutants, A599W, S601W, and T602W, we were unable to evoke functional responses even though the corresponding Ala mutants for these positions expressed well and had Kd(0) values within twofold of the wild type. In an attempt to maximize expression for these nonfunctional mutants, we used an interval of 10 d between recording and injection of 50 ng mRNA with incubation of 10 µM concanavalin A for 1416 min before application of agonist. For wild-type GluR6, these conditions typically gave responses that were in excess of 20 µA at -60 mV after only 4-min application of concanavalin A and saturated the amplifier with longer applications of lectin. The threshold for detection of agonist responses was typically 12 nA at -60 mV. Ramps to -200 mV to check for high affinity polyamine block like that seen for Q590W also failed to reveal latent responses. To distinguish between the possibility that these nonfunctional responses were due to gating mutants, or simply reflected a lack of cell surface expression, oocytes were injected with mRNA for A599W and S601W (T602W was not tested) and stained by indirect immunofluorescence with an antibody directed against the COOH terminus of GluR6. Staining was indistinguishable from that for uninjected oocytes, whereas positive controls with wild-type GluR6 showed intense cell-surface expression as described previously (
Ala and Trp Mutants that Reduce Polyamine Block Cluster on One Face of the KcsA Pore Helix
When the results of the Ala and Trp scans were compared (Fig 2 and Fig 3) it was obvious that the most sensitive regions aligned with the pore helix and selectivity filter of KcsA. When results for the GluR6 sequence from T576 to M589 (which aligns with the pore helix in KcsA) were mapped onto a helical wheel, we found that those mutants which disrupted polyamine block showed a clustered distribution (Fig 4 A). Using this sequence alignment and the KcsA atomic coordinates, we asked where are residues that disrupted polyamine block in GluR6 located in the KcsA structure. This analysis showed that, in the pore helix, the side chains of residues for which Ala and Trp substitution disrupted polyamine block projected either towards the membrane spanning helices and selectivity filter within the same subunit, or towards the selectivity filter of an adjacent subunit (Fig 4 B). In contrast, the amino acid side chains of positions insensitive to substitution with either Ala or Trp projected away from both the selectivity filter and M1 or M2 and likely face lipid. A detailed discussion of results for some individual positions is given later. The other mutants that strongly disrupted polyamine block were either within sequences corresponding to the selectivity filter in KcsA or neutralized the negative charge at E594.
Neutralization of Charged Residues in the Cytoplasmic Linkers between M1 and M2
The results of the Ala and Trp scans revealed strong disruption of polyamine block for mutation of E594 to either alanine or tryptophan. This residue aligns with D80 in KcsA and is located immediately after the selectivity filter near the entrance to the channel (Fig 1). Of note, in cyclic nucleotidegated channels, the mutation E363G, which aligns with E594 and D80, also reduces block by polyamines (
A Glu Scan Maps the Limits of Surface-exposed Residues in the Linker between the Pore Loop and M2
In a sequence alignment of GluR6 and KcsA, E594 corresponds to D80 (Fig 1). In K+ channels, the sequence after D80 links the selectivity filter with the NH2 terminus of the last membrane spanning helix and forms a surface that contributes to the binding site for K+ channel toxins (
These experiments revealed three effects of introducing glutamate in GluR6. First, at six positions in the extracellular surface, a glutamate was able to restore high affinity polyamine block in the E594Q mutant background (Fig 6). Second, in polyamine-free conditions, the weak biphasic rectification produced by the E594Q mutation over the range -100 to -25 mV was also attenuated, such that G-V plots for S593E and K598E recorded in outside-out patches were weakly outward rectifying like those for wild-type GluR6 (not shown). Third, at two positions (P597E and A599E) for which there was no restoration of polyamine block in the E594Q background, the introduction of glutamate in a wild-type background disrupted normal polyamine block (Fig 7).
To obtain a quantitative estimate of changes in the affinity for polyamines resulting from introduction of glutamate at positions surrounding E594, G-V plots were analyzed using a two barrier one site model for permeable block. This revealed that for two positions (L595E and M596E) polyamine block occurred with an affinity only two- to threefold lower than for wild-type GluR6. The Kd(0) values were 3.66 ± 0.04 µM (n = 4) for L595E and 2.39 ± 0.03 µM (n = 7) for M596E versus 1.25 ± 0.02 µM (n = 9) for wild-type GluR6. In previous work with E594Q, in which outside-out patch recording with 1 mM spermine added to the pipet solution was used to increase block and allow estimation of Kd(0) for mutants with low affinity for spermine, we obtained an estimate of 1,020 ± 391 µM (n = 5;
To explore why in the E594Q background, the insertion of glutamate at P597 and A599 failed to restore polyamine block even though the mutation of the surrounding positions to glutamate produced at least a 100-fold increase in polyamine affinity, we repeated the Glu scan in the wild-type background. These experiments also acted as controls for Q590E, Q591E, G592E, S601E, T602E, and R603E, positions for which introduction of glutamate in the E594Q background also failed to restore polyamine block. We found that introduction of glutamate in the wild-type GluR6 background strongly disrupted polyamine block at three of these positions, with little effect at other positions. The mutants showing disruption were P597E, A599E, and T602E (Fig 7). Analysis of G-V plots gave G+80/G-80 ratios of 206 ± 10% (n = 5) for P597E, 75 ± 2.5% (n = 5) for A599E and 206 ± 9.9% (n = 5) for T602E. A comparison of the effects of introducing glutamate in the wild-type and E594Q backgrounds with the results of the Trp scan (Fig 3, Fig 6, and Fig 7) reveals that introduction of Glu and Trp were disruptive at common positions. At A599 and T602, for which the Trp mutants are nonfunctional, introduction of glutamate in the wild-type background disrupted polyamine block. As would be expected, in the E594Q background, introduction of glutamate A599 and T602 failed to restore polyamine block. At P597 the effect of introducing glutamate followed the same qualitative pattern as for A599 and T602; however, the P597W mutant was functional, albeit with a reduction in affinity for spermine (Fig 6). For positions Q590 and Q591, the introduction of glutamate fails to restore polyamine block in the E594Q background (Fig 6) and produces only a small increase in Kd(0) in the wild-type background (Fig 7), indicating that glutamate at these positions does not disrupt channel function.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our experiments used scanning mutagenesis and a simple functional assay, permeable block by cytoplasmic polyamines, to examine secondary structure in the pore of glutamate receptors. The nature of the polyamine binding site(s) in GluRs and the mechanism of block are not yet well understood but were shown previously to be extremely sensitive to mutations at Q590 and E594 (
Interpretation of Mutations in the Pore Helix of K+ Channels and GluRs
Our hypothesis was that the experiments reported here can be interpreted by analogy to previous work on K channels, for which mutations of side chains that are not solvent exposed and that do not directly contact K+ ions were found to disrupt ion selectivity (
Comparable to results for Shaker K+ channels we found that for GluR6 mutation to alanine of W582 and F583 (equivalent to W67 and W68 in KcsA) strongly decreased polyamine block, whereas the mutation F581A had no effect. The aromatic side chains equivalent to W67 and W68 are highly conserved both in other K+ channels and in GluRs, which is consistent with their known structural role in KcsA. Previous work on NMDA receptors had revealed that the mutation W607L in NR2B (equivalent to W67 in KcsA) disrupts Mg2+ block. This effect was interpreted as reflecting a direct interaction of the indole ring with Mg2+ ions via cation- bonding (
The identification of channel lining residues in the NMDA receptor NR1 and NR2 subunits (-helical structure followed by a stretch of consecutively labeled residues. However, when mapped by amino acid sequence alignment to the KcsA structure, this pattern matches extremely well the location of the pore helix and selectivity filter. Thus, just as in the case of cysteine-substituted Shaker K+ channels for which labeling with Ag+ incorrectly assigned pore lining roles to buried aromatic residues (
Molecular Modeling Based on KcsA Coordinates
In the present experiments, particularly in the case of highly disruptive Trp mutations, we observed periodic patterns of changes in polyamine affinity for sequences that aligned with the pore helix of KcsA. When mapped to the structure of KcsA, the sensitive residues face either the selectivity filter or adjacent membrane spanning helices (Fig 4). Using the program O (
A more complex result aided by comparison with the KcsA structure was residue F575 for which mutation to Ala produced a sixfold increase in Kd(0), with a threefold decrease in Kd(0) for the Trp mutant, indicating this position was unlikely to play any major structural role. In KcsA, the equivalent residue I60 is located at the NH2 terminus of the pore helix and contributes to a solvent-exposed surface that binds K+ channel toxins. Consistent with the predicted solvent accessibility of F575 in GluR6, introduction of glutamate was also well tolerated, with a Kd(0) of 0.54 ± 0.01 µM, twofold lower than wild type. In KcsA, the terminal COOH group of a Glu side chain introduced at I60 could be positioned within 12 Å of the COOH group of D80, the equivalent of which in GluR6 is a major determinant of polyamine binding. Perhaps coincidentally, a spermine molecule of length 17 Å could easily span this distance.
We continued using the KcsA structure to help interpret the results obtained for the glutamate scan. As for Trp mutations individual side chains in KcsA were changed to glutamate and a full range of conformers examined. This revealed that side chains that are solvent-exposed and on the surface of KcsA precisely corresponded to positions in GluR6 for which introduction of glutamate was effective in restoring polyamine block in the E594Q background. The C atoms of effective positions were separated by a distance exceeding 17 Å, indicating little positional requirement for negative surface charge (Fig 8). At positions where glutamate was ineffective we observed either of two patterns in the KcsA structure, which corresponded to two distinct patterns for GluR6 responses. For those positions at which introduction of glutamate both failed to restore polyamine block in the E594Q background and was disruptive in the wild-type background, molecular modeling with KcsA revealed that the Glu side chain was not solvent exposed but buried in the protein (Fig 8). This was observed for G77, P83, and T85 in KcsA, which correspond to G592E, P597E, and A599E in GluR6. For T602E, the model was not informative since this side chain (G88 in KcsA), when mutated to Glu, would project into the groove between the outer helix and pore helix, possibly disrupting the packing of M3. For positions where introduction of glutamate failed to restore polyamine block in the E594Q background but was not disruptive in the wild-type background, the mutated side chain was solvent-exposed but either projected towards the inner cavity (Q590E) or was located on the far side of the turret and shielded from the entrance of the pore by surrounding residues (S601E and R603E).
Location and Function of the RNA Editing Site in Glutamate Receptors
The high affinity for polyamines created by the Q590W mutant suggests an unusual molecular mechanism that might give insight into the normal role of the RNA editing site side chain in glutamate receptors. To address this we changed the T75 side chain, which aligns with Q590 in GluR6 to tryptophan. The W75 side chains of individual subunits in the KcsA model were rotated around the CCßC
bonds to minimize bad contacts with other residues. In the resulting structure, shown in Fig 8, the Trp side chains point upwards and away from the selectivity filter and into the central cavity. Remarkably, in the KcsA T75W model, there are relatively few bad contacts between the Trp side chains of adjacent residues provided that they point into the central cavity. Instead, the faces of opposite pairs of indole rings are separated by 8.5 Å, and form a cage that we propose binds aliphatic cation polyamine molecules with high affinity after they pass through the sequence corresponding to the selectivity filter. A related mechanism has been proposed to create the high affinity external TEA binding site in K+ channels (
The results of our study suggest conservation of the pore helix and associated cavity in K+ channels, GluRs and other members of this super family of ion channels. That these channels all have similar structures almost certainly reflects the energetic advantage that this architecture confers when moving ions across the lipid bilayer (
![]() |
Footnotes |
---|
The online version of this article contains supplemental material.
1 Abbreviation used in this paper: GluR, glutamate receptor ion channel.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Drs. P. Seeburg and E. Liman for the gift of plasmids, Ms. E. Tansey for help with immunohistochemical assays, Drs. R. Petralia and R. Wenthold for the gift of GluR6 antibody, Dr. T. Kuner for sharing results before publication, Dr. K. Swartz for numerous discussions and Drs. K. Swartz and C. Cui for comments on the manuscript.
This work was funded by the Intramural Research Program of the National Institutes of Health.
Submitted: 4 January 2001
Revised: 22 February 2001
Accepted: 28 February 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Armstrong, N., Sun, Y., Chen, G.Q., and Gouaux, E. 1998. Structure of a glutamate-receptor ligand-binding core in complex with kainate. Nature. 395:913-917[Medline].
Bähring, R., Bowie, D., Benveniste, M., and Mayer, M.L. 1997. Permeation and block of rat GluR6 glutamate receptor channels by internal and external polyamines. J. Physiol. 502:575-589[Abstract].
Bowie, D., and Mayer, M.L. 1995. Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block. Neuron. 15:453-462[Medline].
Bowie, D., Lange, G.D., and Mayer, M.L. 1998. Activity-dependent modulation of glutamate receptors by polyamines. J. Neurosci. 18:8175-8185
Burnashev, N., Zhou, Z., Neher, E., and Sakmann, B. 1995. Fractional calcium currents through recombinant GluR channels of the NMDA, AMPA and kainate receptor subtypes. J. Physiol. 485:403-418[Abstract].
Burnashev, N., Villarroel, A., and Sakmann, B. 1996. Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues. J. Physiol. 496:165-173[Abstract].
Catterall, W.A. 1995. Structure and function of voltage-gated ion channels. Annu. Rev. Biochem. 64:493-531[Medline].
Chen, G.Q., Cui, C., Mayer, M.L., and Gouaux, E. 1999. Functional characterization of a potassium-selective prokaryotic glutamate receptor. Nature. 402:817-821[Medline].
Cui, C., Bähring, R., and Mayer, M.L. 1998. The role of hydrophobic interactions in binding of polyamines to non NMDA receptor ion channels. Neuropharmacology. 37:1381-1391[Medline].
Doyle, D.A., Cabral, J.M., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., and MacKinnon, R. 1998. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 280:69-77
Egebjerg, J., Bettler, B., Hermans-Borgmeyer, I., and Heinemann, S. 1991. Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature. 351:745-748[Medline].
Everts, I., Petroski, R., Kizelsztein, P., Teichberg, V.I., Heinemann, S.F., and Hollmann, M. 1999. Lectin-induced inhibition of desensitization of the kainate receptor GluR6 depends on the activation state and can be mediated by a single native or ectopic N-linked carbohydrate side chain. J. Neurosci. 19:916-927
Gross, A., and MacKinnon, R. 1996. Agitoxin footprinting the Shaker potassium channel pore. Neuron. 16:399-406[Medline].
Guo, D.L., and Lu, Z. 2000a. Mechanism of cGMP-gated channel block by intracellular polyamines. J. Gen. Physiol. 115:783-797
Guo, D.L., and Lu, Z. 2000b. Mechanism of IRK1 channel block by intracellular polyamines. J. Gen. Physiol. 115:799-814
Heginbotham, L., and MacKinnon, R. 1992. The aromatic binding site for tetraethylammonium ion on potassium channels. Neuron. 8:483-491[Medline].
Heginbotham, L., Lu, Z., Abramson, T., and MacKinnon, R. 1994. Mutations in the K+ channel signature sequence. Biophys. J 66:1061-1067[Abstract].
Hong, K.H., and Miller, C. 2000. The lipidprotein interface of a Shaker K+ channel. J. Gen. Physiol. 115:51-58
Jones, T.A., and Kjeldgaard, M. 1997. Electron-density map interpretation. Methods Enzymol. 277:173-208.
Kashiwagi, K., Pahk, A.J., Masuko, T., Igarashi, K., and Williams, K. 1997. Block and modulation of N-methyl-D-aspartate receptors by polyamines and protons: role of amino acid residues in the transmembrane and pore-forming regions of NR1 and NR2 subunits. Mol. Pharmacol. 52:701-713
Keinänen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, T.A., Sakmann, B., and Seeburg, P.H. 1990. A family of AMPA-selective glutamate receptors. Science. 249:556-560[Medline].
Kraulis, P.J. 1991. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. App. Crystal. 24:946-950.
Kuner, T., Wollmuth, L.P., Karlin, A., Seeburg, P.H., and Sakmann, B. 1996. Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines. Neuron. 17:343-352[Medline].
Kuner, T., Wollmuth, L.P., and Sakmann, B. 1999. The ion-conducting pore of glutamate receptor channels. In Jonas P., Monyer H., eds. Ionotropic Glutamate Receptors in the CNS. Berlin, Germany, Springer Verlag, 219-249.
Li-Smerin, Y., Hackos, D.H., and Swartz, K.J. 2000a. A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel. Neuron. 25:411-423[Medline].
Li-Smerin, Y., Hackos, D.H., and Swartz, K.J. 2000b. -Helical structural elements within the voltage-sensing domains of a K+ channel. J. Gen. Physiol. 115:33-50
Lomeli, H., Wisden, W., Köhler, M., Keinänen, K., Sommer, B., and Seeburg, P.H. 1992. High-affinity kainate and domoate receptors in rat brain. FEBS Lett. 307:139-143[Medline].
Lü, Q., and Miller, C. 1995. Silver as a probe of pore-forming residues in a potassium channel. Science. 268:304-307[Medline].
MacKinnon, R. 1995. Pore loops: an emerging theme in ion channel structure. Neuron. 14:889-892[Medline].
MacKinnon, R., Cohen, S.L., Kuo, A., Lee, A., and Chait, B.T. 1998. Structural conservation in prokaryotic and eukaryotic potassium channels. Science. 280:106-109
Merritt, E.A., and Bacon, D.J. 1997. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277:505-524.
Monks, S.A., Needleman, D.J., and Miller, C. 1999. Helical structure and packing orientation of the S2 segment in the Shaker K+ channel. J. Gen. Physiol. 113:415-423
Ogielska, E.M., and Aldrich, R.W. 1998. A mutation in S6 of Shaker potassium channels decreases the K+ affinity of an ion binding site revealing ionion interactions in the pore. J. Gen. Physiol. 112:243-257
Panchenko, V.A., Glasser, C.R., Partin, K.M., and Mayer, M.L. 1999. Amino acid substitutions in the pore of rat glutamate receptors at sites influencing block by polyamines. J. Physiol. 520:337-357
Parsegian, A. 1969. Energy of an ion crossing a low dielectric membrane: solutions to four relevant electrostatic problems. Nature. 221:844-846[Medline].
Partin, K.M., Patneau, D.K., Winters, C.A., Mayer, M.L., and Buonanno, A. 1993. Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A. Neuron. 11:1069-1082[Medline].
Pearson, W.L., and Nichols, C.G. 1998. Block of the Kir2.1 channel pore by alkylamine analogues of endogenous polyamines. J. Gen. Physiol. 112:351-363
Roux, B., and MacKinnon, R. 1999. The cavity and pore helices in the KcsA K+ channel: electrostatic stabilization of monovalent cations. Science. 285:100-102
Santoro, B., and Tibbs, G.R. 1999. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Annu. NY Acad. Sci. 868:741-764
Schreibmayer, W., Lester, H.A., and Dascal, N. 1994. Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes. Pflügers Arch. 426:453-458.
Schrempf, H., Schmidt, O., Kummerlen, R., Hinnah, S., Muller, D., Betzler, M., Steinkamp, T., and Wagner, R. 1995. A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans. EMBO (Eur. Mol. Biol. Organ.) J. 14:5170-5178[Abstract].
Vyklicky, L., Benveniste, M., and Mayer, M.L. 1990. Modulation of N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones. J. Physiol. 428:313-331[Abstract].
Wenthold, R.J., Trumpy, V.A., Zhu, W.S., and Petralia, R.S. 1994. Biochemical and assembly properties of GluR6 and KA2, two members of the kainate receptor family, determined with subunit-specific antibodies. J. Biol. Chem. 14:1332-1339.
Williams, K., Pahk, A.J., Kashiwagi, K., Masuko, T., Nguyen, N.D., and Igarashi, K. 1998. The selectivity filter of the N-methyl-D-aspartate receptor: a tryptophan residue controls block and permeation by Mg2+. Mol. Pharmacol. 53:933-941
Wo, Z.G., and Oswald, R.E. 1995. Unraveling the modular design of glutamate-gated ion channels. Trends Neurosci. 18:161-168[Medline].
Wood, M.W., VanDongen, H.M., and VanDongen, A.M. 1995. Structural conservation of ion conduction pathways in K channels and glutamate receptors. Proc. Natl. Acad. Sci. USA. 92:4882-4886[Abstract].
Yellen, G. 1999. The bacterial K+ channel structure and its implications for neuronal channels. Curr. Opin. Neurobiol. 9:267-273[Medline].
Zagotta, W.N., and Siegelbaum, S.A. 1996. Structure and function of cyclic nucleotide-gated channels. Annu. Rev. Neurosci. 19:235-263[Medline].