Ion pore properties of ionotropic glutamate receptors are modulated by a transplanted potassium channel selectivity filter

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 33 (2006) 335 – 343

Ion pore properties of ionotropic glutamate receptors are modulated by a transplanted potassium channel selectivity filter Jutta Hoffmann, a Anna Gorodetskaia, b and Michael Hollmann a,⁎ a

Dept. of Biochemistry I–Receptor Biochemistry, Ruhr University Bochum, Building NC, Level 6, Rm. 170, D-44787 Bochum, Germany Institute of Neurobiochemistry, University Witten-Herdecke, D-58448 Witten, Germany

b

Received 1 May 2006; revised 2 August 2006; accepted 17 August 2006 Available online 28 September 2006 The canonical potassium channel selectivity filter motif TVGYG was transplanted into ionotropic glutamate receptors (iGluRs) of the AMPA and NMDA subtype to test whether it renders the iGluRs K+ selective. The TVGYG motif modulated several ion pore properties of AMPA receptor as well as NMDA receptor mutants, e.g., the intraand extracellular polyamine block, current/voltage relationships, open channel block by MK801 and Mg2+, and permeability for divalent cations. However, introduction of the selectivity filter failed to increase the K+ selectivity of homomeric AMPA and heteromeric NMDA receptor complexes, which may be due to absence of selectivity filterstabilizing interaction sites in the iGluR pore domain. Our findings indicate that even if glutamate receptors appear to have the intrinsic capacity for K+ permeability, as is demonstrated by the prokaryotic, glutamate-gated, K+ selective GluR0, the isolated selectivity filter is not able to confer K+ permeability to the relatively unselective iGluR cation pore. © 2006 Elsevier Inc. All rights reserved.

Introduction Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels and mediate the majority of the excitatory neurotransmission in the vertebrate central nervous system. The homo- or heterotetrameric receptor complexes show a subtype-dependent preference for divalent over monovalent cations as well as extraand intracellular polyamine block, which in some subunits is controlled by mRNA editing at the Q/R/N site located at the tip of the membrane-inserted ion pore domain (see Fig. 1A): Q variants of AMPA receptor subunits assemble to form receptor complexes that are permeable for divalent cations and blocked by intra- and extracellular polyamines. In contrast to this, incorporation of R variants into the receptor complex leads to receptors impermeable for divalent cations and insensitive to intra- and extracellular ⁎ Corresponding author. Fax: +49 234 321 4244. E-mail address: [email protected] (M. Hollmann). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2006.08.006

polyamine block (Hume et al., 1991; Swanson et al., 1996, 1997, Verdoorn et al., 1991). In NMDA receptors, permeability for divalent cations similarly is controlled by an asparagine residue (N) at the Q/R/N site (N position), but the amino acid at the adjacent N + 1 position (see Fig. 1B) also influences pore properties like divalent cation permeability and sensitivity for channel blockers (Burnashev et al., 1992; Sakurada et al., 1993; Wollmuth et al., 1998a,b). Rapid repolarization after action potentials as well as regulation of neuronal excitability is mediated by highly potassium-selective K+ channels, which are also homo- or heterotetrameric subunit assemblies. High potassium selectivity is achieved by the highly conserved selectivity filter sequence TVGYG. Each of the four subunits contributes one TVGYG selectivity filter sequence to the pore, with the main chain carbonyl oxygen atoms pointing towards the pore lumen. These carbonyl oxygen atoms form a tube containing four potassium ion binding sites so that potassium ions entering the pore can easily be dehydrated while sodium ions (due to their smaller diameter) cannot (Doyle et al., 1998; Zhou et al., 2001). Interestingly, the pore-forming domains of iGluRs and K+ channels show a similar topology, with both pore regions being organized as membrane-embedded hairpin loops carrying a selectivity filter at the tip of the pore loop (Wo and Oswald, 1994; Hollmann et al., 1994; Wo and Oswald, 1995; Kuner et al., 1996, 2001; and see Fig. 1A). Besides this topological similarity, three amino acids with significant impact on ion channel function – the WGP motif – are conserved between iGluRs and K+ channels (Kuner et al., 2003). These similarities concerning sequence and topology led to the hypothesis that the pore regions of iGluRs and K+ channels may have arisen from a common ancestor. Indeed, in 1999, a potassium-selective, glutamate-gated ion channel protein (GluR0) was discovered in the cyanobacterium Synechocystis, an ion channel which may be regarded as a “missing link” between iGluRs and K+ channels (Chen et al., 1999). Potassium selectivity is conferred upon GluR0 by the canonical potassium channel selectivity filter sequence TVGYG. Thus, glutamate-gated ion channels appear to have the intrinsic capacity for potassium selectivity if provided with the appropriate selectivity filter sequence.

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Fig. 1. Topology and pore domain sequence alignment of ionotropic glutamate receptors and potassium channels. (A) Schematic representation of ionotropic glutamate receptors (left) and voltage-gated potassium channels (right). Glutamate binds at the ligand binding domain (LBD) formed by the S1 and S2 domains (light gray and black). The three transmembrane domains (A, B, C) with the pore domain in between transmembrane domains A and B form the intramembranous part of the receptor. The Q/R/N site is located at the tip of the pore and controls important pore properties such as ion permeability and channel block by, e.g., polyamines or Mg2+. Voltage-gated potassium channels have six transmembrane domains which can roughly be divided in a voltage-sensing module (S1–S4, with S4 carrying positive charges) and a pore module (S5, pore loop, and S6). The pore loop contains a pore helix followed by the selectivity filter sequence TVGYG which generates the extremely high potassium selectivity of potassium channels. (B) Alignment of pore regions of the glutamate receptor wild type and mutant subunits used in this study. N and N + 1 depict the Q/R/N site itself (N) and the amino acid directly following the Q/R/N site (N + 1, nomenclature after Wollmuth et al., 1996). Note that the pore domain of GluR1 is one amino acid shorter than that of the NMDA receptor subunits and thus introduction of the TVGYG selectivity filter sequence “stretches” the AMPA receptor pore by one amino acid.

In this paper, we analyzed the impact of the potassium channel selectivity filter sequence TVGYG on certain pore properties of eukaryotic iGluRs, especially on ion selectivity. We asked the question whether the TVGYG motif renders the eukaryotic iGluR pores potassium-selective. To this end, we introduced the TVGYG motif at the corresponding position into the pore domains of various iGluR subunits representing pharmacologically distinct subtypes of the glutamate receptor family (see Fig. 1B for an alignment of the respective pore regions). We found that the TVGYG motif did not render the iGluRs potassium-selective but that important pore properties such as polyamine block or permeability for divalent cations were influenced by this motif. The potassium channel selectivity filter obviously requires a potassium channel sequence background, providing stabilizing interaction sites for the filter sequence. This suggests that such sites are absent in the iGluR pore domain, resulting in a destabilized and thus non-functional selectivity filter. This conclusion is supported by data from whole pore transplantations between iGluRs and K+ channels (see accompanying paper). With respect to the often cited hypothesis that iGluRs and potassium channels share a common architecture of their pore regions, we conclude on the basis of our data that this similarity does not represent a structural and functional equivalence in every molecular detail but is limited to a general, rather superficial similarity between the pore domains of these two ion channels.

Results and discussion TVGYG mutants of AMPA receptors The AMPA receptor subunit GluR1 carrying the K+ channel selectivity filter sequence TVGYG was expressed in Xenopus oocytes together with cRNA coding for the TAR protein γ2 (stargazin, STG) which is known to increase AMPA receptor surface expression (Chen et al., 2000; Yamazaki et al., 2004; Priel et al., 2005). In the presence of stargazin, 3 days after cRNA injection, robust current responses were obtained upon application of kainate (Fig. 2A). Currents mediated by GluR1(TVGYG) were reduced to 3.1 ± 0.6% of wild type current amplitudes in NFR, and to a similar extent (3.2 ± 0.84%) in KR (see Table 1). In the absence of stargazin, there were only small kainate-induced responses observed in GluR1 (TVGYG)-expressing oocytes, detectable only after a rather long expression period of 12 days or longer (Fig. 2A). Therefore, subsequent analysis of AMPA receptor mutants was always performed in the presence of stargazin. The apparent affinity for kainate was similar for wild type GluR1(Q) and mutant GluR1(TVGYG) (40 ± 4.2 μM kainate for GluR1(Q) and 35 ± 2.6 μM kainate for GluR1(TVGYG), see Fig. 2B). However, the EC50 value for GluR1(Q) obtained here in the presence of stargazin is slightly (∼ 10 μM) lower than that obtained in a previous study without stargazin (Villmann et al., 1999). This is in accordance with new findings indicating that the

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Fig. 2. Functional properties of the AMPA receptor TVGYG mutant. (A) Typical agonist-induced currents recorded in NFR and KR, and in coexpression with the TARP γ2 (STG). (B) Dose–response relationships for glutamate for GluR1(Q) and GluR1(TVGYG). Mean and SEM of 3–4 cells. (C) Representative I/V relationships of GluR1(Q) and GluR1(TVGYG) recorded in NFR in the presence of 150 μM kainate and normalized to the current at − 120 mV (D, E) DNQX and NASP block of GluR1(Q), GluR1(TVGYG), and GluR2(R). 50 μM DNQX and 10 μM NASP were applied in the presence of 150 μM kainate, n = 3–5.

apparent agonist affinity of AMPA receptors is slightly increased in presence of stargazin, probably because of reduced receptor desensitization (Yamazaki et al., 2004; Priel et al., 2005; Turetsky et al., 2005). Similar EC50 values for wild type and mutant indicate that the ligand binding site is not affected by the mutation, a finding that was not unexpected since the mutation is located in the pore domain. However, as the I/V relationships were linear in GluR1(TVGYG) when at the same time GluR1(Q) showed inwardly rectifying I/V curves (Fig. 2C), the TVGYG motif evidently interfered with the binding of internal polyamines in the pore domain which causes inward rectification. Moreover, binding of external polyamine toxins and thus channel block by 1-Naphthylacetyl spermine (NASP) was disturbed in GluR1(TVGYG) while block by the

competitive antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) was not (Figs. 2D and E). In general, inwardly rectifying I/V curves in AMPA receptors carrying a glutamine residue (Q) at the Q/R/N site are caused by voltage-sensitive binding of endogenous intracellular polyamines within the ion pore (Kamboj et al., 1995; Koh et al., 1995; Donevan and Rogawski, 1995). Linear I/V curves similar to those of GluR1(TVGYG) are observed in heteromeric native AMPA receptor complexes containing the GluR2(R) subunit, as well as in homo- or heteromeric recombinant AMPA receptor mutants in which the respective glutamine residue has been changed to arginine (Verdoorn et al., 1991, Hume et al., 1991; Herlitze et al., 1993; Blaschke et al., 1993; Washburn and Dingledine, 1996). Similarly, block by extracellularly applied polyamine toxins is

Table 1 Functional properties of TVGYG mutants of the AMPA receptor GluR1 and the NMDA receptor complex Subunit combination

GluR1(Q) GluR1(TVGYG) NR1-1a + NR2B NR1-1a-TVGYG + NR2B NR1-1a + NR2B-TVGYG

NFR

KR

MgR

Absolute current [nA]

Relative current [%]

n

Absolute current [nA]

Relative current [%]

n

INFR/IKR

n

Absolute current [nA]

928.7 ± 171 33.3 ± 4.7 1047.5 ± 278 3.1 ± 1.4 102.2 ± 10

– 3.1 ± 0.6 – 0.3 ± 0.45 9.8 ± 3.1

3 3 15 8 8

1268.3 ± 330 46 ± 11 960.4 ± 304 2.2 ± 1.4 94.6 ± 6.5

– 3.2 ± 0.84 – 0.23 ± 0.43 9.9 ± 2.1

6 6 7 15 7

0.77 ± 0.06 0.74 ± 0.08 1.79 ± 0.45 1.53 ± 0.23 1.44 ± 0.35

3 3 7 7 7

n.d. 243 ± 75 3.6 ± 1.75 214.6 ± 9.6

Relative current [%]

n

– 1.5 ± 1.1 88 ± 6.1

10 8 8

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controlled by the amino acid at the Q/R/N site such that Q variants are blocked by extracellular polyamine toxins while R variants are not (Blaschke et al., 1993; Herlitze et al., 1993; Washburn and Dingledine, 1996). The dependence of intra- and extracellular polyamine block on the amino acid present at the Q/R/N site is explained by the interaction between the amino acid at the Q/R/N site and a conserved aspartate residue (D) located deeper in the pore. This aspartate residue is also known to influence intra- and extracellular polyamine block and might be regarded as a second binding site for polyamines (see Fig. 1B and Dingledine et al., 1992; Washburn and Dingledine, 1996). It is suggested that a salt bridge forms between the positively charged arginine residue and the acidic carboxyl group of the aspartate residue so that both binding sites are disrupted. However, the threonine residue present in GluR1 (TVGYG) has a polar side chain similar to that of glutamine, and, due to extension of the pore by one amino acid (which increases the distance between the Q/R/N site and the aspartate residue), it seems unlikely that such a masking effect can explain the reduced internal and external polyamine block in GluR1(TVGYG). We instead suggest that introduction of the TVGYG selectivity filter into the pore domain of GluR1 has led to an overall structural perturbance of the pore domain and thus internal as well as external polyamine binding sites have been disrupted. Next, we tested the K+ permeability of GluR1(Q) in comparison to that of GluR1(TVGYG). In a first approach, we compared current amplitudes in NFR (low potassium concentration) and in KR (high potassium concentration) (Fig. 2A, left panel). In a second approach, we determined reversal potentials according to Chen et al. (1999) using Ringer’s solutions containing 10, 50, and 115 mM extracellular K+ with Na+ used to maintain constant ionic strength (Figs. 3A and B). We used such mixtures of K+ and Na+ rather than testing K+ alone (and balancing ionic strength by adding NMDG) since current amplitudes decrease substantially when one of the major permeable ions is removed completely. This would be counterproductive since current amplitudes of the AMPA receptor TVGYG mutant are already small in NFR (see Table 1) and robust current responses larger than 10 nA are required for recording reliable I/V relationships. In both experimental setups, no differences in potassium permeability were detected, leading to the conclusion that the TVGYG motif does not render AMPA receptors potassium-selective but influences some important pore properties such as internal and external polyamine block. No influence of the TARP stargazin itself on K+ permeability of AMPA receptors could be detected as evidenced by absence of a shift in reversal potentials (data not shown). TVGYG mutants of NMDA receptors Next, we analyzed TVGYG mutants of NMDA receptors, the pore of which is one amino acid longer compared to non-NMDA receptors (see Fig. 1B). Consequently, introduction of the TVGYG motif did not lead to extension of the pore by one amino acid. Since native NMDA receptors are heteromers comprised of NR1 and NR2 subunits (Ishii et al., 1993), the TVGYG motif was introduced into NR1-1a as well as into NR2B. Coexpression of either NR1-1a(TVGYG) + NR2B or NR1-1a + NR2B(TVGYG) led to inward currents which were not blocked by Mg2+ ions (Fig. 4A, MgR). I/V relationships therefore lacked the NMDA receptor-typical Mg2+ block, but instead were rather linear at negative membrane potentials (Fig. 4B). The TVGYG

Fig. 3. Comparison of potassium permeability of wild type GluR1(Q) and GluR1(TVGYG) GluR1(TVGYG). (A) Ratio of current amplitudes in NFR and KR. Mean and SEM of 3–5 cells. (B) Reversal potentials in Ringer's solutions with varying ratios of K+ and Na+ determined by recording of I/V relationships in the presence of 150 μM kainate. Mean and SEM of 6–7 cells.

mutation obviously has similar effects as have mutations of the Q/ R/N site (N position) or the following position (N + 1 position) which are known to influence the Mg2+ block, especially in the NR2B subunit (Burnashev et al., 1992; Kupper et al., 1996; Wollmuth et al., 1998a, b). Coexpression of both mutant subunits (NR1-1a(TVGYG) + NR2B(TVGYG)) did not result in any agonist-induced currents, nor did expression of NR1-1a(TVGYG) or NR2B(TVGYG) alone (data not shown). Since the coexpression of NR1-1a(TVGYG) and NR2B resulted in quite small current responses (3.1 ± 1.4% of wild type in NFR, see Table 1), the following analyses concentrate on the influence of the NR2B(TVGYG) mutant on functional properties of the NMDA receptor complex. Presence of NR2B (TVGYG) reduced the current amplitudes to approximately 10% of those of wild type (see Table 1). The apparent glutamate affinity was not changed significantly (Mann–Whitney test, see Fig. 4C), and comparable EC50 values for glutamate of 1.47 ± 0.03 (NR1-1a + NR2B) and 1.74 ± 0.01 (NR1-1a + NR2B(TVGYG)) were obtained. As was the case for the AMPA receptor TVGYG mutants, similar apparent agonist affinities suggest that the ligand binding domain remains unaffected by the mutations which is introduced in the pore region. However, functional ion pore properties determined by the Q/R/ N site were indeed changed since NR2B(TVGYG)-containing receptors showed an IC50 for the open channel blocker MK801 which was increased by 300-fold compared to wild type NMDA receptor complexes. Nevertheless, complete block could still be achieved given appropriate MK801 concentrations were used (Figs. 4D, E). Point mutations of the Q/R/N site (N position) and the following amino acid (N + 1 position) led to a similar decrease in MK801 sensitivity (Mori et al., 1992; Kashiwagi et al., 2002).

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Fig. 4. Functional properties of TVGYG mutants of NMDA receptors. (A) Representative current responses of oocytes expressing wild type or mutant subunit combinations in NFR, KR, and MgR. (B) Representative I/V relationships of the subunit combinations NR1-1a + NR2B (1), NR1-1a(TVGYG) + NR2B (2), and NR1-1a + NR2B(TVGYG) (3). The currents were normalized to the current recorded at − 120 mV. Inset: enlarged view for better resolution. (C) Dose– response relationship for glutamate for the subunit combinations NR1-1a + NR2B and NR1-1a + NR2B(TVGYG). Recordings were performed in the presence of 10 μM glycine. Mean and SEM of 3 cells each. (D) Dose–response relationship for inhibition by MK801 for the subunit combinations NR1-1a + NR2B and NR1-1a + NR2B(TVGYG). Currents elicited by 300 μM glutamate and 10 μM glycine were recorded at − 70 mV and blocked by increasing concentrations of MK801 (n = 3). The EC50 values for glutamate and the IC50 values for MK801 are given in panel E.

As was already indicated (Figs. 4A, B), the Mg2+ block was altered in NMDA receptor complexes containing the NR2B (TVGYG) mutant subunit. A careful analysis of the Mg2+ block revealed that, surprisingly, the IC50 for Mg2+ measured at −70 mV was not drastically changed (Fig. 5A and Table 2). Interestingly, the residual current in the presence of a saturating Mg2+ concentration of 10 mM was greatly enhanced when the NMDA receptor complex contained NR2B(TVGYG) (Fig. 5B and Table 2). The high amount of residual current in the presence of 10 mM Mg2+ suggested that the TVGYG selectivity filter motif led to an increased Mg2+ permeability of the receptor complex containing NR2B(TVGYG). Since the agonist-induced currents of the NR11a + NR2B(TVGYG) subunit combination in most cases were hardly large enough to record I/V relationships in NFR, the NR11b subunit was used instead. This subunit does not show protoninduced inhibition at physiological pH and should give rise to 50% larger currents than the NR1-1a subunit (Traynelis and Cull-Candy,

1990; 1991). However, even when current amplitudes were large enough to record I/V relationships in NFR, they were far to small for recording them in 8 mM MgR, in which 8 mM Mg2+ is the only (putatively) permeable cation (data not shown). Therefore, instead of trying to detect a shift in the reversal potential, Mg2+ permeability was tested by comparing the amplitudes of agonistinduced currents in the aforementioned 8 mM MgR and in NMDG Ringer (NMDG-R), in which no external cation was present. Both wild type NMDA receptors and those containing the subunit NR2B (TVGYG) showed inward currents in NFR (Fig. 5C, left panel). Wild type NMDA receptors showed small outward currents in NMDG-R which were presumably carried by K+ ions leaving the cell, while in 8 mM MgR, no current was detected (Figs. 5C, D). In the presence of the NR2B(TVGYG) subunit, outward currents were recorded in NMDG-R and, interestingly, inward currents were detected in 8 mM MgR, indicating an increased Mg2+ permeability in NR2B(TVGYG)-containing NMDA receptor complexes (Figs. 5C, D). In the TVGYG mutant of the NR2B

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Fig. 5. Mg2+ block of TVGYG mutants of NMDA receptors. (A) Dose–response relationship for Mg2+ and (B) amount of residual current in the presence of 10 mM Mg2+ for the subunit combinations NR1-1a + NR2B, NR1-1a + NR2B(TVGYG), NR1-1b + NR2B, and NR1-1b + NR2B(TVGYG). Currents elicited by 300 μM glutamate and 10 μM glycine were recorded at − 70 mV and blocked by increasing concentrations of Mg2+ (n = 6–7). (C, D) Analysis of Mg2+ permeability of NMDA receptors containing NR2B(TVGYG). (C) Representative current responses in NFR (control condition), NMDG-R (containing no external cations except 1 μM MgCl2), and 8 mM MgR (containing only Mg2+ as external cation) in various Ringer's solutions for the subunit combinations NR11b + NR2B and NR1-1b + NR2B(TVGYG). Note that there is a net inward current in oocytes expressing NR1-1b + NR2B(TVGYG) in 8 mM MgR. Quantification of these data is given in (D), where the ratio Ix/INFR was calculated, Ix standing for the current amplitude in NMDG-R and 8 mM MgR, respectively (n = 7–10).

subunit, the N position is changed from asparagine to threonine, and the asparagine at the N + 1 position is mutated to valine. Mutations at the N + 1 position of the NR2 subunit similar to that investigated here have been shown to have a high impact on Mg2+ permeability, as demonstrated by an increased Mg2+ permeability of recombinant NMDA receptors containing glycine, serine, or glutamine instead of asparagine either at the N or N + 1 position of the NR2 subunit (Williams et al., 1998, Wollmuth et al., 1998a, b). Even if native NMDA receptors are slightly permeable for Mg2+ in the absence of other ions (Stout et al., 1996), currents carried by Mg2+ are only detected in high extracellular Mg2+ (70 mM) where the electrochemical gradient driving Mg2+ to enter the cell is 10× larger than in the 8 mM MgR used here. Therefore, the intrinsic Mg2+ permeability does not interfere with the

additional Mg2+ permeability seen in NMDA receptor complexes containing NR2B(TVGYG). On the other hand, the incomplete Mg2+ block may have arisen from a combination of an increased Mg2+ permeability and a Mg2+induced current potentiation since NMDA receptor-mediated responses are enlarged by extracellular Mg2+ ions dependent on subunit combination (Paoletti et al., 1995). This potentiation is mediated by the NR2B subunit and prevented by NR1-1b splice variants and may explain the fact that the amount of residual current in presence of the NR1-1b subunit is nearly 2× smaller than in the presence of the NR1-1a subunit (Fig. 5B). Besides its critical role for Mg2+ block and open channel block by MK801, the Q/R/N site (N position) and the position following the Q/R/N site are critical for the permeability for divalent cations.

Table 2 Properties of Mg2+ block of NMDA receptors containing the mutant NR2B(TVGYG) subunit Subunit combination

IC50 [μM]

Hill

Residual current [%]

n

NR1-1a + NR2B NR1-1a(TVGYG) + NR2B NR1-1b + NR2B NR1-1b(TVGYG) + NR2B

41.1 ± 1.2 42.6 ± 1.5 37.4 ± 1.1 86.8 ± 1.6

− 0.92 ± 0.15 − 0.89 ± 0.29 − 1.12 ± 0.12 − 0.93 ± 0.20

0.9 ± 0.5 41.1 ± 4.4 1.5 ± 0.9 26.5 ± 3.9

7 6 3 3

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For NR1 subunits, the N position is most important (Wollmuth et al., 1996; Williams et al., 1998; Kashiwagi et al., 2002), whereas in NR2 subunits the N + 1 and N + 2 positions have a larger impact on divalent cation permeability (Wollmuth et al., 1996). Therefore, we checked whether the TVGYG selectivity filter sequence influenced the Ba2+ permeability of the NMDA receptor complex, by recording agonist-induced currents in NaR, which contained mainly Na+ as the permeable cations, and comparing the currents with those obtained in BaR, which contained mainly Ba2+ (see Experimental procedures for details). To maximize current amplitudes, in these experiments, the NR1-3b subunit was used, which is known to yield the largest responses of all NR1 splice variants in oocytes (Schmidt et al., 2006). Current amplitudes in BaR were greatly reduced in NR2B(TVGYG)-containing NMDA receptors, while the opposite was observed in wild type NMDA receptors, indicating that Ba2+ permeability is indeed reduced (Figs. 6A, B). Introduction of the TVGYG motif into NR2B changes the asparagine residue at the N + 1 position to a valine and the serine residue at the N + 2 position to a glycine. Similarly, a decreased permeability for Ba2+ and Ca2+ was observed in NMDA receptors containing mutant NR2A or NR2B subunits which contained glycine either at the N + 1 position or at the N + 2 position (Wollmuth et al., 1996, Williams et al., 1998). To test if permeability for K+ was also changed in NR2B (TVGYG)-containing receptors, we again compared current amplitudes in NFR and KR, but found no difference in mean current amplitudes (Fig. 6C). To analyze the K+ permeability more carefully, we determined the reversal potentials in Ringer’s

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solutions with increasing K+ concentrations in which no other ion but a small amount of Ca2+ was present for membrane stability (see Experimental procedures section for details). The shift of reversal potentials was nearly identical for wild type NMDA receptors and NMDA receptors containing the NR2B (TVGYG) mutant subunit, suggesting that permeability for monovalent cations is not changed by the potassium channel selectivity filter sequence TVGYG (Fig. 6D). Taken together, our data indicate that introducing the potassium channel selectivity filter at corresponding positions in the relatively unselective, cation-permeable AMPA and NMDA receptor pore domains does not confer an increased or otherwise changed K+ permeability on the respective iGluR mutants. In case of the NMDA receptor complexes, permeability for K+ could only be determined in heteromeric complexes, most probably containing two NR2B (TVGYG) and two wild type NR1 subunits. Lack of an increased K+ permeability might therefore be attributed to the fact that there were only two instead of four selectivity filter sequences lining the pore. In AMPA receptors composed of GluR1(TVGYG), however, each subunit possesses one TVGYG motif and, thus, four selectivity filter sequences line the pore in GluR1(TVGYG). In principle, iGluRs do have the intrinsic capacity for K+ selectivity as exemplified by the potassium-selective prokaryotic glutamate receptor GluR0 (Chen et al., 1999). In this receptor, the entire pore-forming unit (TMD A, pore loop and TMD B) is indeed functional when transplanted into the eukaryotic sequence background of the kainate receptor subunit GluR6 (see accompanying paper). The chimera adopted some electrophysiological properties of the pore donor, such as a linear I/V relationship and presumably some potassium selectivity.

Fig. 6. Ba2+ and K+ permeability of TVGYG mutants of NMDA receptors. (A, B) Ba2+ permeability of NR2B(TVGYG)-containing NMDA receptors. (A) Representative agonist-induced currents recorded in NaR (containing mainly Na+ ions) and in BaR (containing only Ba2+ ions). (B) Ratios of currents recorded in NaR and BaR, mean and SEM of 5 cells. (C) Comparison of current amplitudes in NFR and KR (mean and SEM of 5–7 cells). (D) Reversal potentials in Ringer's solutions with varying K+ concentrations and no other cation present. NMDG was added to balance osmolarity. I/V relationships were recorded in the presence of 300 μM glutamate and 10 μM glycine (mean and SEM of 3–5 cells).

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This demonstrates that the entire pore-forming unit of the prokaryotic, potassium-selective GluR0 can be gated by the gating machinery of a eukaryotic GluR, meaning that, in general, eukaryotic GluRs have an intrinsic capacity for potassium selectivity. Nevertheless, introducing only the selectivity filter into an AMPA as well as into an NMDA receptor sequence background does not result in exclusively potassium-permeable ion pores. We therefore assume that structurally important interaction sites in the potassium channel pore helix and adjacent transmembrane domains (Doyle et al., 1998; Zhou et al., 2001) are absent in the iGluR pore domain. Thus, in the mutants analyzed here, the TVGYG selectivity filter sequence might have been destabilized and kept in a conformation inappropriate for conveying K+ selectivity on the iGluR pore. These results imply that the ion pore domains of iGluRs and potassium channels despite some general similarities cannot replace each other in determining ion selectivities. The same conclusion was also drawn on the basis of the pore transplantation data presented in the accompanying paper. Our observations lead us to the conclusion that eukaryotic iGluR pores and potassium channel pores are not (or not any longer) of high enough structural similarity to allow functional pore exchange or exchange of functional determinants such as the selectivity filter transplantation. Experimental Methods Mutagenesis Mutagenesis of the iGluR pore region was performed by overlap extension PCR in which the products of two independent PCRs carrying the desired mutations plus an overlapping region of approximately 18 bp length at their ends and beginnings, respectively, were combined in a third PCR. After initial hybridization of the compatible regions and fill-in, the resulting fragments served as templates and were amplified by adding upstream and downstream primers. These fragments were subcloned into cDNAs coding for the respective rat iGluR subunits GluR1, GluR6, NR1-1a, and NR2B. All mutant constructs were sequenced across the PCR-generated regions using the dideoxynucleotide chain termination method (Sanger et al., 1977). The vector used for all constructs was pSGEM, which is a modified version of pGEMHE (Liman et al., 1992) carrying the multiple cloning site of pBluescript (Stratagene, Heidelberg, Germany) and two additional linearization sites, SphI and NdeI (Everts et al., 1997).

20 Hz with a low pass filter. Data were digitized with a sampling rate of 200 Hz using an A/D converter (ITC16 Computer interface, Instrutech Corp., Long Island, NY, USA) and analyzed with Pulse software (Heka, Lambrecht, Germany). Recordings were performed in normal frog Ringer's solution (NFR, containing in mM: 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 HEPES–NaOH, pH 7.2) and in high potassium Ringer's solution (KR, containing in mM: 115 KCl, 1.8 CaCl2, 10 HEPES–NaOH, pH 7.2). Ca2+ in NFR was replaced by Mg2+ when Mg2+ block was tested. This Ringer's solution was called MgR and contained (in mM): 115 NaCl, 2.5 KCl, 1.8 MgCl2, 10 HEPES–NaOH, pH 7.2. Potassium permeability of AMPA receptor mutants was tested in K+/ Na+ Ringer's solutions containing 1.8 mM MgCl2 and 10 mM HEPES, plus different ratios of K+ to Na+ ions which were (K+/Na+, in mM): 10/105, 50/ 65, and 115/0. To test for NMDG and Mg2+ permeability of NMDA receptors, NMDG Ringer (containing 116 mM NMDG, 10 mM HEPES, 1 μM MgCl2) and 8 mM MgR (containing 108.6 mM NMDG, 10 mM HEPES, 8 mM MgCl2) were used. To test for K+ permeability, K+/NMDG Ringer's solutions were used which contained 10 mM HEPES and 0.2 mM CaCl2, as well as 2.5, 50, or 115 mM KCl, with appropriate amounts of NMDG to preserve osmotic pressure. For NMDA receptors, permeability for divalent cations was analyzed using sodium Ringer's solution (NaR, containing in mM: 64 NaCl, 2 KCl, 1 BaCl2, 10 HEPES, 64 NMDG) and barium Ringer's solution (BaR, containing in mM: 64 BaCl2, 2 KCl, 10 HEPES). The pH of all Ringer's solutions was 7.2 and adjusted with HCl and NMDG, respectively. Whenever ionic permeabilities of NMDA receptors were analyzed in the presence of Ca2+, oocytes were pre-injected with 50 nl 50 mM EGTA to abolish endogenous Ca2+-induced Cl− conductances. As an alternative, Ba2+ or a low concentration of Mg2+, which was found insufficient to block wild type NMDA receptors, was used as divalent cation (see Fig. 5A). The glutamatergic agonists L-glutamate (Glu), kainic acid (KA), and the NMDA receptor co-agonist glycine (Gly) were prepared in the respective Ringer's solution in concentrations of 300 μM (Glu), 150 μM (KA) and 10 μM (Gly). They were applied for 20 s. Electrodes were filled with 3 mM KCl and had resistances between 0.5 and 1 MΩ. All AMPA receptor constructs were coexpressed with the rat TAR protein γ2 (stargazin) at a 10:1 ratio to increase surface expression and AMPA receptor-mediated currents. Current–voltage (I/V) relationships for glutamate receptor constructs were determined with 2 s voltage ramps from − 150 mV to +50 mV in the presence of 300 μM glutamate. I/V curves were also obtained without ligand to subtract background conductances from the GluR-mediated current.

Acknowledgments cRNA synthesis cRNA synthesis was performed as described earlier (Hollmann et al., 1994). Briefly, template cDNA was linearized with a suitable restriction enzyme, and cRNA was prepared from 1 μg of linearized cDNA using an in vitro transcription kit (Fermentas, St. Leon-Rot, Germany). Each of the nucleotides was used at 800 μM, except for GTP (200 μM); 400 μM m7 GpppG was included for capping. The reaction time was extended to 4 h using T7 RNA polymerase. Radioactive trace labeling was performed with [32P]UTP (Amersham, Braunschweig, Germany) to allow calculation of yields and transcript quality checks by agarose gel electrophoresis.

This work was supported by the Graduiertenkolleg 736 “Development and Plasticity of the Nervous System” of the Deutsche Forschungsgemeinschaft. We would like to thank Sabine Kott, Department of Biochemistry I — Receptor Biochemistry, Ruhr University Bochum, for the cDNA of γ2 and Dr. Herrmann Pusch, Department of Cell Physiology, Ruhr University Bochum, for invaluable help with the ion selectivity measurements.

Electrophysiological recordings

References

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