Structural similarities between glutamate receptor channels and K(+) channels examined by scanning mutagenesis

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 S593-L600 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.

Figure 1

Homology between glutamate receptors and K⁺ channels. (A) Amino acid sequence alignment of pore region residues for GluR6, GluRD, GluR0, and KcsA. Residues common to KcsA and any of the three glutamate receptors are shown in bold; yellow shaded boxes indicate conservative substitutions; a red arrow indicates the conserved negative charge at E594. The original transmembrane helix assignments for GluR6 that were based on hydropathy analysis are shown as the top set of shaded boxes labeled “M1,” “M2,” and “M3” (Egebjerg et al. 1991; Lomeli et al. 1992). The numbering for GluR6 assumes processing of a 31–amino acid signal peptide. The dashed line below the GluR6 sequence indicates the extent of the alanine and tryptophan scans performed in the present study. Deletions in the sequence for GluR6, GluRD, and GluR0 are indicated by an asterisk. Below the sequence of KcsA shaded boxes indicate α-helical regions in the X-ray diffraction structure (Doyle et al. 1998); the numbering is for full-length protein. B and C show the secondary structure of KcsA with the sequence corresponding to that used for the GluR6 alanine and tryptophan scans shaded lighter than the surrounding structure; black and red spheres indicate the Cα positions of residues T75 and D80 in KcsA that align with Q590 and E594 in GluR6. The pore helix is indicated by P, and the membrane spanning helices by M1 and M2; this and subsequent figures were drawn using Molscript (Kraulis 1991) and Raster3D (Merritt and Bacon 1997). In B, the orientation with respect to the membrane is that expected for glutamate receptors and is rotated by 180° with respect to that for K⁺ channels. In C, rotation by 90° shows the cytoplasmic surface and entrance to the pore for the orientation found in glutamate receptors.

Figure 2

Alanine scanning mutagenesis disrupts polyamine block with characteristic patterns separating sensitive and insensitive residues. (A) Leak-subtracted I-V plots reveal reduced rectification and increased outward currents for Q590A but not M589A. (B) Mean G-V plots for these mutants with the SD plotted at 20-mV intervals. Responses for wild-type GluR6 (dashed line) nearly overlie responses for M589A. (C) K _d(0) values for alanine-substituted GluR6 mutants from D567 to R603 measured using Boltzmann fits to G-V plots like those shown in B. Shaded boxes indicate responses for which G-V plots did not show the biphasic rectification characteristic of polyamine block; a conservative lower estimate of K _d(0) for these positions is >100 μM. The shaded horizontal box indicates a plus/minus twofold change from the K _d(0) value for wild-type GluR6. α-Helical regions in the secondary structure of KcsA mapped onto the GluR6 sequence as shown in Fig. 1 are shown above the bar plot.

Figure 3

Tryptophan scanning mutagenesis strongly disrupts polyamine block. (A) Mean G-V plots for the adjacent pair of mutants M589W (n = 3) and Q590W (n = 6); dashed lines show the fit of one (Q590W) or two (M589W) Boltzmann functions; SDs are plotted every 20 mV. (B) Mean G-V plots for S580W and G584W, two examples of mutants for which introduction of tryptophan abolished the strongly voltage-dependent rectification resulting from permeable block by polyamines. (C) Changes in K _d(0) for tryptophan-substituted GluR6 mutants from D567 to R603 measured from responses like those shown in A and B; shaded boxes indicate responses like those for G584W and S580W for which G-V plots did not show the biphasic rectification characteristic of polyamine block. Of the 37 positions studied, no response (NR) was recorded for 3 positions. The shaded horizontal box indicates a plus/minus twofold change from the K _d(0) value for wild-type GluR6. α-Helical regions in the secondary structure of KcsA mapped onto the GluR6 sequence as shown in Fig. 1 are shown above the bar plot.

Figure 4

Results of scanning mutagenesis suggest a pore helix in GluR6. (A) Helical wheel analysis of Ala and Trp substitutions from T576 to M589. Black circles indicate positions for which substitution of tryptophan abolished polyamine block (Fig. 3 C, shaded bars). White circles indicate positions for which alanine substitution increased K _d(0) to more than four times the value for wild-type GluR6 as shown in Fig. 2 C. Shaded circles indicate positions for which both Ala and Trp were effective. (B) Secondary structure for two adjacent KcsA subunits viewed along the axis of the pore helix (P) for the subunit on the left; the orientation matches that shown for the helical wheel plot; M1 and M2 indicate the outer and inner membrane spanning helices; an arrow indicates the central axis of the permeation pathway and points towards the central cavity (Fig. 1). Side chains are drawn in black for KcsA positions aligning with GluR6 residues mutation of which strongly disrupts polyamine block.

Figure 5

Neutralization of charged amino acids in the linkers between the pore helix and transmembrane helices. (A) Membrane folding model for GluR6 based on the alignments shown in Fig. 1 indicates the positions of individual residues for which glutamate and aspartate residues were neutralized one at a time by a mutation to glutamine and asparagine. M1, M2, and M3 indicate the three membrane spanning segments in GluRs; P indicates the pore helix. Scissors indicate positions at which deletion mutants were generated by introduction of a stop codon. Three basic residues were also neutralized by mutation of lysine and arginine side chains to asparagine and glutamine, respectively. (B) Plots of the ratio of conductance at +80/−80 mV for the charge neutralization and deletion mutants shown in A; the shaded box indicates the 95% confidence interval for wild-type responses. Of the 13 Glu and Asp positions tested using charge neutralization, only E594 was required for high affinity polyamine block.

Figure 6

Reintroduction of glutamate into the linker between the pore loop and second transmembrane helix restores high affinity polyamine block in GluR6 E594Q mutants. (A) Mean G-V plots for L595E and M596E examples of E594Q double mutants showing restoration of polyamine block. Data points plotted every 20 mV show the mean ± SD of responses for 4–5 oocytes per mutant. The dashed line shows responses for wild-type GluR6; and a dotted line indicates responses for the charge neutralization point mutant E594Q. (B) Mean values for K _d(0) measured from Boltzmann fits to responses like those for L595E and M596E. The shaded background for P597 and A599 shows a linear interpolation of K _d(0) values from E594 (wild type) to L600E and emphasize that, at these positions, reintroduction of glutamate in the E594Q background fails to restore high affinity polyamine block. Note that P597 and A599 are flanked by residues for which introduction of glutamate was highly effective. α-Helical regions in the secondary structure of KcsA mapped onto the GluR6 sequence as shown in Fig. 1 are shown above the bar plot.

Figure 7

Disruption of GluR6 function by introduction of glutamate near the start of the second membrane spanning helix. (A) Mean G-V plots for P597E, A599E, and T602E in the wild-type GluR6 background. Data points plotted every 20 mV show the mean ± SD of responses for 5 oocytes per mutant. The dashed line shows the response for wild-type GluR6. (B) Changes in K _d(0) for Glu and Trp point mutants; the shaded horizontal box indicates a plus/minus twofold change from the K _d(0) value for wild-type GluR6. Mutants that failed to give functional responses are indicated by NR. Note that for A599 and T602, introduction of glutamate disrupted polyamine block, whereas introduction of tryptophan produced nonfunctional receptors. α-Helical regions in the secondary structure of KcsA mapped onto the GluR6 sequence (Fig. 1) are shown above the bar plot.

Figure 8

Mapping GluR6 Q/R site and glutamate scan mutations to KcsA secondary structure. Two complete KcsA subunits plus the pore helix and surrounding linkers to M1 and M2 for a third subunit are shown and labeled as for Fig. 1. The side chain for T75 that maps to Q590 in an alignment with GluR6 (Fig. 1) was mutated to tryptophan. The side chain bond angles between the Cα–Cβ–Cγ atoms were adjusted to rotate the indole ring into the cavity of KcsA; the resulting aromatic cage has minimal bad VDW contacts with surrounding residues. The Cα positions of residues in the pore surface that, when mutated to glutamate, restored high affinity polyamine block in the E594Q background are shown as red spheres. The two positions for which mutation to glutamate abolished polyamine block in the wild-type background are shown as black spheres.