Additional Point Mutations

In addition to the point mutations in the FDLSL motif, three other amino acids within the linker A were selected for mutation. The selection was based on dierences between delta1 and delta2, since the linker A transplantation from the two subunits had such distinct eects on GluR1 properties. Two positions were selected that were identical in delta1 and GluR1, but showed a non-conservative change in delta2: K507T and F513C. The former position is located in the rst part of the linker (EKTVD), the latter in the middle part (MFACLAP). In addition, glycine 509 in GluR1 was mutated to aspartic acid, which is present at this position in delta2. In delta1, a polar serine is located at the corresponding site. Hence, the negatively charged aspartic acid in delta2 was judged to be a candidate amino acid of suciently distinct character to be involved in inducing the observed dierences upon linker A transplantation from either delta1 or delta2.

Expression of the G509D mutant in Xenopus oocytes revealed a mutant receptor that was non-responsive to both glutamate or kainate application (Fig. 5.45, Tab. 5.12). A potential reason for this non-responsiveness is the close positioning of the negatively charged aspartic acid (G509D) to the positively charged lysine (K507) in the mutant.

In addition, the proline (P508) positioned right between G509D and K507 might infer a conformation that is incompatible with function when two oppositely charged amino acids reside in neighboring positions. To investigate this possibility, a double GluR1 mutant was generated in which in addition to the G509D mutation K507 was mutated to threonine (T538 in delta2). The resulting mutant GluR1-(K507T, G509D) was analyzed as well.

Table 5.12: Glutamate- (300 µM) and kainate-evoked (150 µM) current responses, current ratios, and average leak current of the GluR1 linker A mutants: GluR1-(F513C), GluR1-(G509D), GluR1-(K507T), and GluR1-(K507T, G509D). Wild type values are listed for comparison.

Construct IGlu[nA] (n) IKA[nA] (n) Mean leak [nA] (n) IGlu/IKA (n)

GluR1(Q)op 43 ± 8 (17) 388 ± 39 (17) 114 ± 18 (17) 0.10 ± 0.01 (17) K507T 5 ± 2 (13) 8 ± 1 (13) 227 ± 62 (13) 0.50 ± 0.16 (11)

G509D 0 ± 0 (6) 0 ± 0 (6) 93 ± 30 (6) −

K507T, G509D 19 ± 11 (11) 57 ± 7 (12) 362 ± 96 (12) 0.27 ± 0.12 (11) F513C 3 ± 1 (12) 8 ± 1 (11) 258 ± 54 (12) 0.36 ± 0.07 (11)

Figure 5.45: Representative cur- rent responses of the indicated GluR1 point mutants to 300 µM Glu (upper row), or 150 µM KA (lower row). Note that the point mutation of both K507T and G509D in one construct yields a responsive mutant, while the sole mutation of G509D renders the re- spective mutant unresponsive.

All four mutants K507T, G509D, K507T+G509D, and F513C were expressed in oocytes, and current responses to glutamate and kainate application recorded. Except for the G509D mutant (see above), all other mutants displayed small current responses (Tab. 5.12). While kainate reliably evoked responses above the detection level, glutamate re- sponses could not be recorded from every oocyte tested. Hence, although the three mutants were functional, current responses were too small and dependent on a well- expressing batch of oocytes for further characterization. Interestingly, the generation of the K507T+G509D double mutant was successful in producing weakly functional chan- nels. This implies that the introduction or deletion of charged amino acid side chains at this particular site in the linker can be decisive for functionality in oocytes.

Control of surface expression Almost all linker A mutations had a substantial im- pact on current amplitudes. Apart from altered electrophysiological properties this can also be due to increased or decreased surface expression. Therefore, surface expression

was controlled by western blot analysis (Fig. 5.46). Expression of all generated linker A mutants was examined in the same experiment to allow a comparison of expression levels. The blot shows that all mutants were present in the plasma membrane. No gross dif- ferences in surface expression levels were detected, although the expression of the E520S point mutant was reduced to some extent. Dierences in surface expression were overall minimal, while expression on the level of the whole cell showed clear dierences in this ex- periment. Weaker whole-cell expression for the FDLS, EKTVD+FDLSL, K507T+G509D and E520S mutants was detected in comparison to wild type. Although all samples were prepared simultaneously from oocytes obtained from the same frog, reliable quantitative interpretation would require corroboration by several repetitions of the experiment. Since the weaker expression was minimal for the surface fractions, and was by no means re- stricted to only mutants that had displayed small current responses, the experiment was not repeated. The analysis of surface expression did not support altered expression lev- els as the reason for the substantial dierences in current amplitudes of the individual linker A mutants.

Figure 5.46: Western blot analysis of whole-cell (T) and surface (S) protein fractions (14-20 oocytes) of the indicated GluR1 linker A mutants. Respective molecular weights are given in [kDa]. For comparison, the full linker A mutant (right) and the wild type GluR1 (left) were analyzed alongside. Note that all mutants are expressed in the plasma membrane. Surface-expression was detected for all mutants. However, expression of the E520S mutant was slightly reduced.

Desensitization of I521L The conservative mutation of I521 to leucine in GluR1 in- creased current amplitudes in the Xenopus oocyte system roughly 10-fold for glutamate- induced responses and 4-fold for kainate-induced responses. Increased current amplitudes

in oocytes might be due to increased surface expression, reduced desensitization, slower gating kinetics, higher single channel conductance, higher open probabilities or any com- bination of these eects. Since no indication of increased surface expression was detected in Western blot analysis, desensitization kinetics of the mutant were analyzed in HEK293 cells (Fig. 5.47).

Figure 5.47: Analysis of desensitization of the GluR1- (I521L) mutant in HEK293 cells. Representative responses of whole-cell patches to application of Glu (3 mM) or KA (600 µM) recorded from HEK293 cells. Black bars indicate the duration of agonist application.

However, no signicant dierence between wild type and mutant were detected in extent of desensitization and desensitization time constants. Both wild type and the I521L mutant desensitized completely upon application of glutamate. Desensitization time constants were 6.1 ± 0.7 ms (n = 7) for the wild type and 7.0 ± 0.9 ms (n = 7) for the I521L mutant. Hence, the observed increase in current amplitudes in the oocyte system could not have been due to slower or less pronounced desensitization.

In conclusion, the described mutagenesis of the GluR1 linker connecting the S1 domain with TMD A showed that this region of the AMPA receptor is highly sensitive to muta- tion. The introduced lurcher characteristics upon transplantation of the full linker A of delta2 to GluR1 can be primarily attributed to the mutation of the FDLSL motif. These ve amino acids are right adjacent to TMD A, and their simultaneous mutation was suf- cient to mimic all major characteristics of lurcher. However, lurcher properties were less pronounced, and qualitative dierences as seen with the response to CNQX surfaced.

Notably, the point mutation of the last amino acid before TMD A substantially in- creased current amplitudes in the oocyte system. This increase was neither due to in- creased expression levels nor to less or slower desensitization. Most interesting was the detection of small responses to the sole application of CNQX or NBQX of mutant recep- tors with point mutations in the FDLSL motif. This is especially intriguing with regard to the recent nding that TARPs can infer responsiveness to CNQX and DNQX to wild type AMPA receptors (Menuz et al., 2007).