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Indeed, all generated AMPA receptor chimeras were capable of forming functional recep- tors, regardless whether only S1, only S2, or both parts of the LBD were exchanged for the corresponding parts of GluR6. Contrary to this nding, prior generation of AMPA and kainate receptor chimeras between GluR3 and GluR6 had yielded a non-functional AMPA receptor as soon as the S2 domain from GluR6 was swapped (Stern-Bach et al., 1994). However, since no crystal structures had been available at the time, the borders of the exchanged segments had been placed quite dierently from the ones that were chosen in the present study. While the S1 domain exchange in the study by Stern-Bach et al. had included S1 and linker A, the S2 exchange had included TMD B, linker B, S2, linker C, and TMD C. Therefore, the reported non-functionality of the S2-containing chimeras likely originated from the inclusion of the kainate receptor TMDs and linkers, which were not transplanted in the present study.

Since both S1 and S2 constructs form functional chimeras, this set of constructs could be used to decipher which subtype-typical properties are conferred by S1 and which by S2. In this regard, the available crystal structures for the LBD of GluR2 (Armstrong et al., 1998) and GluR6 (Nanao et al., 2005) provide an excellent structural framework for the interpretation of the observed electrophysiological properties.

Reduced desensitization paired with low agonist potency The S1 transplantation resulted in a particular interesting combination of receptor properties: characterization of the chimera strongly suggests that it is less-desensitizing, while it displays a 60-fold reduction in glutamate potency. Both phenomena have not been reported for an AMPA receptor mutant in that particular combination. However, from looking at the structural data both eects can be explained as follows:

First, the swapping of S1 encompasses the change of leucine 479 to tyrosine, a point mutation which individually blocks desensitization (Stern-Bach et al., 1998). Hence, it is not surprising, that the AMPA receptor with the S1 domain of GluR6 shows properties indicative of reduced desensitization. Indeed, this point mutation was originally discovered by the swapping of S1 (Stern-Bach et al., 1998). Crystal structures show that the amino acid at position 479 in S1 interacts with residues from S2, and thereby inuences the stability of the dimer interface between the LBDs of two adjacent subunits (Sun et al., 2002). Analogous to the action of CTZ on AMPA receptors, a tyrosine at this position stabilizes the interface and prevents desensitization (Armstrong and Gouaux, 2000; Jin et al., 2005). This strongly suggests that the S1 chimera is less-desensitizing due to a stabilization of the dimer interface between the GluR6-derived tyrosine in S1 with the S2 domain from GluR1.

Second, it has been shown that agonist potency is inuenced by the strenght of a cross- lobal interface that forms upon agonist-induced cleft closure between the two lobes (D1 and D2) of the LBD (Robert et al., 2005; Weston et al., 2006a; Maier et al., 2007). In the process, the agonist is locked between D1 and D2, and the stability of this closed-bound form decisively governs deactivation, recovery from desensitization, and agonist potency. Although both S1 and S2 each contribute a part of the two lobes D1 and D2, this cross- lobal interface is mostly formed between residues from S1 (D1 side) and S2 (D2 side). Exchanging S1 or S2 thus completely swaps one side of the cross-lobal interface. The observed low agonist potency strongly argues for a considerable weakening of these cross- lobal interactions, which would destabilize the closed-bound conformation of the chimera, and thereby reduce agonist potency. A second line of evidence for such a destabilization is based on the increased potency of the competitive antagonist CNQX. The less stable the agonist is locked between D1 and D2, the easier CNQX can replace it.

Interestingly, the GluR3 chimera that carried the S1 domain plus linker A from GluR6 did show reduced desensitization, but no reduction in glutamate potency (Stern-Bach et al., 1994). If the low potency of the GluR1-GluR6 chimera was indeed due to a desta- bilization of cross-lobal interactions, this should also have been the case for the GluR3- GluR6 chimera. However, glutamate potency for the GluR3-GluR6 S1 chimera did not deviate from the respective wild type values (Stern-Bach et al., 1994). Three dierences in the compared chimeras might account for this discrepancy: the usage of GluR3 instead of GluR1 (20 aa changes, mostly conservative), the usage of a ip instead of a op isoform (9 aa changes), or the inclusion of linker A (6 aa changes). Since the eect on glutamate potency was quite substantial, it would be interesting to elucidate which of the three dierences is responsible - assuming that it is not a cumulative eect of all three.

S2 decisively inuences the agonist prole The rank order of agonist potency at GluR1 receptors is quisqualate > AMPA > domoate > glutamate > kainate. By

contrast, for GluR6 receptors it is domoate > kainate > quisqualate > glutamate, while AMPA does not activate homomeric GluR6 (Stern-Bach et al., 1994; Swanson et al., 1997). Although agonist potencies were not determined for all agonists, and thus potency rank orders cannot be compared, the amplitudes of domoate-elicited responses provided a good indicator for a switch in prole. Clearly, the S2 domain of GluR6 was decisive in switching an AMPA- to a kainate-like agonist prole. The applied concentration of domoate elicited the largest amplitudes at chimeras containing the S2 domain of GluR6.

Likewise, AMPA insensitivity was conferred by the S2 domain of GluR6. This is in agreement with the report that mutation of the residue N721 in GluR6 to serine confers AMPA sensitivity to GluR6 (Swanson et al., 1997). In AMPA receptors, the corresponding position is occupied by threonine (T686) which is permissive for the large degree of domain closure induced by AMPA binding. In the closed-bound conformation, this threonine forms a polar contact with a glutamate (E402) residue from S1 (Armstrong et al., 1998). In GluR6, N721 forms a polar contact with E440 from S1 (Nanao et al., 2005). Although the exact mechanism remains to be determined, the asparagine in GluR6 might prevent the LBD from adopting the high degree of domain closure that is necessary for AMPA binding (Nanao et al., 2005). Since the swapping of S2 encompasses the change of threonine 686 to asparagine, it is not surprising that the AMPA receptor with the S2 domain of GluR6 is insensitive to AMPA.

Sensitivity to lectin modulation Kainate receptor desensitization is potently inhib- ited by treatment with the lectin Concanavalin A, while AMPA receptors are only very mildly aected (Everts et al., 1997). The eect of the lectin is critically dependent on N-linked glycosylation of at least one of the nine extracellular glycosylation sites of GluR6 (Everts et al., 1999). The GluR1 LBD chimeras show manipulation by the lectin only if the S2 domain of GluR6 was transplanted. The potentiating eect, however, could not be determined for the S2 chimera, since this was only responsive after treatment with the lectin. For the AMPA receptor that carried the full LBD of GluR6, glutamate responses were 10-fold increased after lectin treatment, whereas responses of wild type GluR6 re- ceptors were increased 2000-fold. The fact that the S1 transplantation yields a construct whose responses are insensitive to lectin treatment is in agreement with the nding that responses of the non-desensitizing GluR1-(L479Y) mutant are also not increased by the treatment (Thalhammer et al., 2002). Since GluR1-(L479Y) and the S1 chimera are pre- sumably non-desensitizing, a treatment that targets receptor desensitization is not likely to have any further eect.

One could expect that swapping the complete LBD of GluR6 confers the high suscep- tibility to Concanavalin A to the AMPA receptor chimera. However, this is not observed. This nding might be an indication that the dierent susceptibility of AMPA and kainate receptors to lectin modulation is a result of the subtypes' particular arrangement of LBD

and NTD, and not solely attributable to special characteristics of the GluR6 LBD. Alter- natively, it cannot be excluded that the LBD exchange has some inuence on the general structure of the domain assembly and thereby reduces the eect of lectin treatment.

Notably, the three chimeras oer the possibility to examine the specic contributions of S1 vs S2 to the eect of AMPA receptor-specic allosteric modulators such as CTZ (Armstrong and Gouaux, 2000; Jin et al., 2005) or the kainate receptor-specic require- ment for certain extracellular ionic species (Bowie, 2002; Paternain et al., 2003; Plested and Mayer, 2007) - experiments which were not within the scope of this thesis.

The GluR6 LBD gates AMPA receptors Contrary to the ndings of Stern-Bach et al., the complete transplantation of the LBD of GluR6 yielded a functional AMPA receptor chimera. This discrepancy is most likely due to the dierent sizes of the transplanted segments. The inclusion of TMD B and TMD C in previous transplantations likely had a pronounced eect on the ion pore conformation and might have disrupted channel function. It is interesting that the specic combination of an AMPA receptor-derived TMD A and a kainate receptor-derived TMD C always resulted in non-functional chimeras, while the reverse combination is compatible with channel function (Stern-Bach et al., 1994). The above described x-large pore constructs between GluR1 and GluR6 nicely t this pattern of functionality. Moreover, the functionality of the GluR1-GluR6 LBD chimera proves that as long as TMD A and C are derived from an AMPA receptor, functionality is preserved.

In view of the proposed gating model, these chimeras provide an additional line of evidence that TMD A and C interaction is both of pivotal importance for channel func- tion and obviously distinct between AMPA and kainate receptors. With regard to another AMPA receptor specicity - the modulation by TARPs - the observed pattern of function- ality calls for a chimeric AMPA receptor that carries both TMD A and TMD C (including the respective linkers) derived from GluR6.

Although AMPA and kainate receptors are often regarded as close cousins, the genera- tion of chimeric receptors has proven to be an extremely valuable tool for the elucidation of structurally distinct features. So far, all of these features govern either the stabil- ity of the dimer interface between the LBDs of two adjacent subunits or the stability of cross-lobal interactions within the LBD of one subunit. If the proposed third region of interactions between TMD A and TMD C really exists, chimeras might again prove valuable to elucidate its dimensions and subtype specicity.