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The transplantations described above strongly suggest, that it is the LBD which harbors the key to understand why delta receptors are non-functional in heterologous expression systems. Ionotropic function in wild type delta receptors might be regulated such that the function of the LBD is inhibited in its native environment. To investigate this, the GluR6 LBD was exchanged for that of delta2 in a second set of LBD transplantations.

Oocytes expressing either of the resulting three chimeras GluR6-(S1)delta2, GluR6- (S2)delta2, and GluR6-(S1+S2)delta2 were tested for responses to glutamate and kainate application after ConA treatment (Tab. 5.23). No current responses could be detected.

Table 5.23: Average responses of GluR6-delta2 S1 and S2 chimeras to glutamate (300 µM), or kainate (150 µM) after ConA treatment recorded in NFR. GluR6(Q) was used as positive control.

Construct IGlu[nA] (n) IKA [nA] (n)

GluR6-(S1)delta2 0 ± 0 (5) 0 ± 0 (5) GluR6-(S2)delta2 0 ± 0 (3) 0 ± 0 (3) GluR6-(S1+S2)delta2 0 ± 0 (7) 0 ± 0 (6) GluR6(Q) 3 922 ± 647 (8) 4 457 ± 665 (7)

Figure 5.69: Representative current responses of delta1, delta2, GluR6-(S1+S2)delta2, and GluR6(Q) to glycine (500 µM) and D-serine (500 µM). Both agonists were applied in the presence and absence of glutamate (300 µM). GluR6(Q) served as positive, uninjected oocytes as negative control (n = 2-6).

However, this non-responsiveness to glutamatergic agonists was not surprising given the recent publication of the delta2 LBD structure, which showed that glycine and D-serine bind to delta2 (Naur et al., 2007). Consequently, glycine and D-serine were tested on

GluR6-(S1+S2)delta2 in the presence and absence of glutamate (Fig. 5.69). To control the expression level, GluR6(Q) wild type was used as positive control. Uninjected oocytes served as negative control, since other amino acids than glutamate can induce small, unspecic responses in oocytes. Since binding of glycine and D-serine was reported for delta2, both delta1 and delta2 wild types were included in this experiment. The small responses of delta1 to glycine and D-serine were unspecic, since they were also recorded from uninjected oocytes. None of the tested receptor-ligand combination showed clear receptor-mediated current responses. Hence, although glycine and D-serine bind to delta2 and reduce the spontaneous current through delta2-lurcher channels (Naur et al., 2007), neither substance evokes current responses at delta wild type receptors or the GluR6- (S1+S2)delta2 chimera.

Assuming that the in vivo function of delta receptors is not merely metabotropic but also ionotropic, there are basically two reasons that could account for their electrophysi- ological silence in heterologous expression systems: Either essential cofactors or auxiliary proteins indispensable for ionotropic function are absent in the expression systems, or the receptors have some intrinsic property that prevents their proper activation and is not yet understood. The experiments described in this thesis were designed to thoroughly investigate the latter possibility.

Within the scope of this thesis the two delta receptor subunits were systematically analyzed by reciprocal domain transplantation. Intrinsic ion channel properties of iGluRs are mainly determined by three essential domains: the LBD, the ion pore, and the linkers that connect the two. Each of these domains were exchanged for those of selected func- tional iGluR subunits. The chosen approach should allow to determine intrinsic properties of single delta receptor domains, or alternatively show which part of the delta receptors needs to be exchanged to yield agonist-responsive ion channels. Towards this end, a total of 22 linker chimeras, 7 ion pore chimeras, and 11 ligand binding domain chimeras were constructed and carefully analyzed for function in Xenopus oocytes.

Following some general remarks on domain transplantation, the results described are discussed according to the transplanted domain of the receptors. Since unexpected and interesting eects of certain linker transplantations on AMPA receptor gating spured additional extensive mutational studies within the linker preceding TMD A, a large part of this discussion focuses on AMPA receptor gating. The general importance of this work with respect to ionotropic function of the delta receptor subunits is discussed last.

6.1 Domain Transplantation

Previously, domain transplantations were used to discern subtype-specic receptor prop- erties of AMPA and kainate receptors, such as assembly (Stern-Bach et al., 1994; Ayalon and Stern-Bach, 2001; Ayalon et al., 2005), desensitization (Stern-Bach et al., 1998), or binding of allosteric modulators (Balannik et al., 2005). However, the technique proved also useful to establish basic functionality of single domains derived from otherwise non- functional receptors (Villmann et al., 1997, 1999; Strutz-Seebohm et al., 2003). Here, domain transplantation was applied to both delta receptors for the latter purpose.

The premise for any new insight into delta receptor function was the construction of functional chimeric receptors, a task which represented the greatest challenge. To maximize the chances of obtaining functional chimeras, the exchange partners for the delta receptors were selected such that they were the best possible compromise between the closest relative and those iGluR subunits that give large responses in oocytes.

Presumably, the functionality of a chimeric subunit is decisively inuenced by the se- lection of the domain borders for transplantation. If the borders are placed in the middle of a functionally important structural motif (such as the dimer interface of two LBDs), this may thwart the production of a functional chimera. In the least, it restricts the conservation of function for exchange partners for which this motif is highly conserved and thus remains intact despite its disruption. Preservation of ionotropic function in chimeras derived from only distantly related exchange partners was hence a likely indica- tion that a complete functional module was exchanged. Non-functional chimeras derived from very closely related subunits, on the other hand, were indicative for the disruption of a functional module and thus provided information about where not to place borders for transplantation cassettes. Therefore, control chimeras between two homomerically func- tional iGluR subunits, the AMPA receptor GluR1 and the kainate receptor GluR6, were always constructed rst. The relative distance between GluR1 and GluR6 subunits (40%) is considerably lower than that between delta and AMPA or kainate receptors (∼65%). Still, the less was known about a functional module and its respective borders, the more risky remained its transplantation.

Planning chimeric receptors was thus a tightrope walk between selecting suitable do- main borders and exchange partners while trying to analyze subunits with unknown func- tion. One drawback of the technique is that it can never be excluded that domain trans- plantation alters the general structure of the protein. However, if the technique yielded functional chimeric receptors, this in itself proved functional integrity of the exchanged part of the two parent subunits.