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Comparative Study
. 2007 Jun 20;27(25):6800-9.
doi: 10.1523/JNEUROSCI.0284-07.2007.

Na+/Cl- Dipole Couples Agonist Binding to Kainate Receptor Activation

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Free PMC article
Comparative Study

Na+/Cl- Dipole Couples Agonist Binding to Kainate Receptor Activation

Adrian Y C Wong et al. J Neurosci. .
Free PMC article

Abstract

Kainate-selective ionotropic glutamate receptors (GluRs) require external Na+ and Cl- as well as the neurotransmitter L-glutamate for activation. Although, external anions and cations apparently coactivate kainate receptors (KARs) in an identical manner, it has yet to be established how ions of opposite charge achieve this. An additional complication is that KARs are subject to other forms of cation modulation via extracellular acidification (i.e., protons) and divalent ions. Consequently, other cation species may compete with Na+ to regulate the time KARs remain in the open state. Here we designed experiments to unravel how external ions regulate GluR6 KARs. We show that GluR6 kinetics are unaffected by alterations in physiological pH but that divalent and alkali metal ions compete to determine the time course of KAR channel activity. Additionally, Na+ and Cl- ions coactivate GluR6 receptors by establishing a dipole, accounting for their common effect on KARs. Using charged amino acids as tethered ions, we further demonstrate that the docking order is fixed with cations binding first, followed by anions. Together, our findings identify the dipole as a novel gating feature that couples neurotransmitter binding to KAR activation.

Figures

Figure 1.
Figure 1.
Allosteric modulation of GluR6 kainate receptors. a, Membrane currents evoked by 10 mm Glu (Vm of −20 mV; patch 05714p1) in the presence of 150 mm NaCl, 2 mm Ca2+, and 1 mm Mg2+ (Control). Replacement of NaCl with CsCl, NaNO3, the removal of divalent ions, and external acidification (pH 5.5) are shown in the same patch recording. b, Summary plots of the experiment shown in a comparing the effect of ion modulation on the amplitude (black bars) and decay kinetics (white bars) of KARs. Data are expressed as the mean ± SEM; n = 5 patches for each condition. *p < 0.05, significant difference from control (Student's two-tailed, paired t test).
Figure 2.
Figure 2.
The M770K mutation delineates between channel amplitude and decay kinetics. a, Top, Sequence alignment for selected regions of the S1 and S2 subunits for AMPA and KARs, with the 770 position highlighted in yellow. Positive and negative residues are identified in blue and red, respectively. Note that the S1 and S2 domains are located in disparate parts of the primary sequence. Bottom left, Crystal structure (Protein Data Bank number 1S50) of the region surrounding M770 drawn using PyMol (DeLano Scientific, Palo Alto, CA) showing the interaction between the S1 and S2 domains. Note a nest of positive and negatively charged amino acids exists directly adjacent to M770 that may constitute an cation-binding site. Bottom right, Detailed view of M770 shows the close proximity of a number of exposed carbonyl oxygen atoms that may also represent putative Na+ binding sites. b, The effect of the M770K mutation on KAR modulation by external cations (Cs+) and anions (NO3; patch 041008p3). c, Left, Normalized raw data traces (from b) showing that M770K abolishes the effect of ions on decay kinetics. The right shows the response profile of GluR6M770K for an extended series of anions and cations. There is no change in decay kinetics for all ions tested (top right), but response amplitude is still modulated (bottom right). Data are mean ± SEM for at least six patches in each ionic condition for both GluR6wt and GluR6M770K.
Figure 3.
Figure 3.
pH modulation of GluR6wt and GluR6M770K. a, Raw data showing the effect of lowering pH (i.e., increasing H+ concentration) on wild-type GluR6 receptors (patch 04615p3). b, The effect of lowering pH on GluR6M770K (patch 06525p1). External acidification leads to a decrease in amplitude but no change in the decay kinetics of both GluR6wt and GluR6M770K. c, Pooled data for an extended pH range for GluR6wt (black squares) and GluR6M770K (white circles). Dose–response curves were best fitted with a single binding site isotherm, with Imax constrained to 100%. The IC50 was pH 6 for GluR6wt, and pH 6.8 was for GluR6M770K. Hill slopes (nH) were 1 and 0.7, respectively. d, The effect of pH on channel decay kinetics. Data were fitted with a linear fit, and there was no significant change in decay kinetics with decreasing pH in both wild-type (black squares) and mutant (white circles) receptors. Data are mean ± SEM of at least six patches for each pH.
Figure 4.
Figure 4.
Ca2+ modulation of GluR6wt and GluR6M770K. a, Raw data showing the effect of increasing external Ca2+ on wild-type GluR6 KARs (patch 050920p1). b, The effect of Ca2+ on GluR6M770K (patch 06601p1). Insets to a and b show normalized raw data traces emphasizing an acceleration of decay kinetics with increasing [Ca2+]o in GluR6wt (inset, a) but not with GluR6M770K (inset, b). c, Dose–inhibition curve for Ca2+ for wild-type and M770K mutant KARs. Data were fit with a single binding site isotherm with a steady-state amplitude (Io, 22% of peak), with Imax constrained to 100%. The IC50 for Ca2+ was 1 mm for GluR6wt and 2 mm for GluR6M770K. The Hill slopes (nH) were 1.3 and 1.0, respectively. d, The acceleration in decay kinetics observed in GluR6wt with increasing Ca2+ concentration is abolished by M770K. Data are mean ± SEM of at least five patches at each calcium concentration.
Figure 5.
Figure 5.
Possible mechanisms for monovalent ion interactions at KARs. a, Schematic showing three distinct models to explain the effect of monovalent anions and cations on GluR6 KARs. b, Top, Crystal structure showing critical amino acid residues that constitute the proposed anion binding site (Protein Data Bank number 2F34). Although only one dimer is shown, the ion is also conjugated by the corresponding amino acids from the adjacent subunit. b, Bottom, Proposed anion binding site containing point mutations (R775K, D776E, and T779N) that interfere with both anion and cation modulation of GluR KARs. Note that each mutation elicits an enlargement of the anion binding pocket through changes in the orientation of each residue. c, The effect of changing external cation species with the various anion binding site mutants. Both R775K and T779N abolish cation modulation, whereas cation modulation is markedly reduced in the D776E mutant. Data are mean ± SEM of at least three patches per mutant in each cation.
Figure 6.
Figure 6.
A basic amino acid at the M770 position eliminates both anion and cation effects on KARs. a, Two basic (Lys and Arg; patch 041008p3 and patch 06520p3), a noncharged (Ala; patch 051202p3), and an acidic amino acid mutation (Glu; patch 06411p2) were made at Met770 to assess whether changes in charge or side chain length preferentially affected modulation by anions or cations. The raw data traces (GluR6wt; patch 04116p2) show the effect of external cation substitutions from Na+ to K+ and Cs+. Note that basic amino acid mutations lead to a substantial acceleration of decay kinetics in all ions tested. b, Pooled data for cation (top) and anion (bottom) substitutions with the various GluR6M770 point mutations. Addition of a positively charged amino acid (Lys or Arg) at this position abolishes both cation and anion modulation. However, external ions still modulate KARs with an aliphatic (Ala) or acidic (Glu) amino acid in place of Met770. Data are mean ± SEM of at least five patches for each mutation in each ionic condition.
Figure 7.
Figure 7.
Basic amino acids at the M770 position act as surrogate cations. a, Left, Raw data traces showing anion substitutions in which Na+ is the major cation (patch 04130p2). There is an acceleration of decay kinetics when Br is replaced with Cl and NO3. Right trace shows the same experiment as left but with Rb+ as the major cation (patch 06209p1). Note that anion substitutions do not change decay kinetics in the presence of Rb+. b, An extended series of anion substitutions with either Na+ (black bars) or Rb+ (white bars) as the dominant cation. There is no change in decay kinetics when anions are substituted in Rb+ in contrast to Na+. Data are mean ± SEM for at least six patches in each anion with Na+ or Rb+ as the major cation. c, The effect of changing the external Na+ (filled squares) and Rb+ (open circles) concentrations on the decay kinetics of wild-type GluR6 (left) and GluR6M770K (right) receptors. Note that the decay kinetics GluR6wt matches that of GluR6M770K only when Rb+ ions are used.
Figure 8.
Figure 8.
A positively charged residue at the M770 position abolishes the absolute requirement for ions. a, Superimposed family of membrane currents evoked by 1 mm l-Glu between −100 and +110 mV in Na+-free solutions. KARs with Met770 substituted with either an aliphatic (M770A; patch 06724p3) or an acidic (M770D; patch 06727p2) amino acid still have an absolute requirement for external ions. In contrast, substitution of Met770 with a positively charged amino acid (Arg; patch 06713p2) results in an outward current in Na+-free solutions (middle). b, Mean current–voltage plots from at least three experiments in physiological Na+ (150 mm; open triangles) and Na+-free (0 mm; filled circles) solution, demonstrating that GluR6M770R is functional, whereas GluR6M770A and GluR6M770D are nonfunctional.

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