Western blot analysis of GABA A receptors labeled on the surface of HEK cells. HEK cells were cotransfected with ␣ 1 , ␤ 3 , and ␥ 2 subunits or with ␣ 1 *, ␤ 3 , and ␥ 2 subunits. GABA A receptors expressed on the cell surface were immunolabeled by adding ␣ 1 (1 – 9) antibodies to intact cells, and were then extracted, immunoprecipitated, and analyzed by SDS-PAGE and Western blots using digoxygenized ␣ 1 (1 – 9), ␤ 3 (1 – 13), or ␥ 2 (1 – 33) antibodies. 

Contexts in source publication

Context 1

... be suitable for this purpose because it is homologous to ␣ 1 (1 – 117) but could not be coprecipitated with ␥ 2 subunits (or ␤ 3 subunits) after coexpres- sion in HEK cells (Fig. 2). To incorporate binding sites of the ␣ 1 subunit, several chimeras were constructed by replacing the C-terminal part of the ␤ 3 (1 – 115) fragment with the correspond- ing ␣ 1 sequences (Fig. 2). These chimeras were transfected into HEK cells together with full-length ␥ 2 subunits. Expressed sub- units were precipitated from cell extracts with ␥ 2 (319 – 366) anti- bodies. The precipitate was subjected to SDS-PAGE, and the proteins were detected with digoxygenized ␤ 3 (1 – 13) antibodies in Western blots. The actual expression of the chimeras was con- fi rmed by precipitation and detection with ␤ 3 (1 – 13) antibodies (data not shown). In ␤ 3 (1 – 115)chim ␣ 1 (101 – 117) the 17 C-terminal amino acids of the ␤ 3 (1 – 115) fragment were replaced by amino acids 101 – 117 of the ␣ subunit. As indicated in Figure 2, this chimera could not be coprecipitated with full-length ␥ 2 subunits from appropriately cotransfected HEK cells, demonstrating the speci fi city of the ␥ 2 (319 – 366) antibodies used and indicating that amino acids ␣ 1 (101 – 117) are not able to induce binding to ␥ 2 subunits. In ␤ 3 (1 – 115)chim ␣ 1 (80 – 117), the amino acid sequence ␤ 3 (78 – 115) was replaced by ␣ 1 (80 – 117). This construct was able to bind to full-length ␥ 2 subunits (Fig. 2), but not to full-length ␤ 3 subunits (data not shown). Because amino acids ␣ 1 (101 – 117) were not suf fi cient to induce binding to ␥ 2 subunits as discussed above, this indicated that amino acids 80 – 100 of the ␣ 1 subunit are important for binding to ␥ 2 subunits. To directly con fi rm this conclusion, the construct ␤ 3 (1 – 115)chim ␣ 1 (80 – 100) was generated (Fig. 2), in which amino acids ␣ 1 (80 – 100) were incorporated into ␤ 3 (1 – 115), replacing amino acids ␤ 3 (78 – 98). As expected, this chimera was able to bind to ␥ 2 subunits. To investigate which part of the ␣ 1 (80 – 100) sequence is re- sponsible for binding to ␥ 2 subunits, four additional chimeras were constructed. In ␤ 3 (1 – 115)chim ␣ 1 (80 – 86), amino acids ␤ 3 (78 – 84) were replaced by the amino acids ␣ 1 (80 – 86), in ␤ 3 (1 – 115)chim ␣ 1 (87 – 93) the sequence ␤ 3 (85 – 91) was replaced by ␣ 1 (87 – 93), in ␤ 3 (1 – 115)chim ␣ 1 (94 – 100) the sequence ␤ 3 (92 – 98) was replaced by amino acids ␣ 1 (94 – 100), and in ␤ 3 (1 – 115)chim ␣ 1 (80 – 93) the sequence ␤ 3 (78 – 91) was replaced by ␣ 1 (80 – 93) in the ␤ 3 (1 – 115) fragment. None of these chimeras was able to bind to ␥ 2 subunits. These results indicate that the whole ␣ (80 – 100) sequence is necessary for binding to ␥ subunits. To investigate the importance of the ␣ 1 (80 – 100) sequence not only for the assembly of truncated subunits and dimers, but also for assembly of full-length subunits and pentameric receptors, a full-length ␣ 1 chimera ( ␣ 1 *) was constructed in which the se- quence ␣ 1 (79 – 100) was replaced by the sequence ␤ 3 (77 – 98). The additional exchange of the amino acid 79 of the ␣ 1 subunit in ␣ 1 * was necessary to avoid the generation of two adjacent prolines that could have destroyed the conformation of the resulting chi- mera (Fig. 2). In control experiments, it was demonstrated that the extent of expression of the ␣ 1 * chimera was similar to that of the ␣ 1 subunit in HEK cells (data not shown). HEK cells were then cotransfected with ␣ 1*, ␤ 3 , and ␥ 2 sub- units and subunits expressed were investigated by immuno fl uo- rescence and confocal laser microscopy. As shown in Figure 3, ␣ 1 * (Fig. 3 A ) and ␤ 3 subunits (Fig. 3 B ) could be easily detected on the surface of intact cells, but for the ␥ 2 subunit only a weak labeling was observed (Fig. 3 C ), although the labeling of the ␥ 2 subunit in permeabilized cells (Fig. 3 F ) was comparable with that of ␣ 1 (Fig. 3 D ) and ␤ 3 (Fig. 3 E ) subunits. In HEK cells cotrans- fected with ␣ 1 , ␤ 3 , and ␥ 2 subunits, all three subunits could be detected on the cell surface (Fig. 3 G – I ). Because previous results have indicated that ␣ 1 subunits alone in contrast to ␣ 1 ␤ 3 subunit combinations do not form receptors that are incorporated into the plasma membrane to a signi fi cant extent, these results sug- gested that ␣ 1 * predominantly formed receptors with ␤ 3 subunits that are expressed on the cell surface, but the ability to form receptors containing ␥ 2 subunits was signi fi cantly reduced. To quantify this phenomenon, HEK cells were cotransfected with ␣ 1 , ␤ 3 , and ␥ 2 subunits or with ␣ 1 *, ␤ 3 , and ␥ 2 subunits. GABA A receptors expressed on the surface of the cells were labeled by an incubation of intact cells with ␣ 1 (1 – 9) antibodies. Antibody labeled receptors were extracted and precipitated by addition of Immunoprecipitin. The precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized ␣ 1 (1 – 9) antibodies (Fig. 4). In contrast to ␣ 1 subunits (51 kDa, the weak 46 kDa band presumably represents a degradation product), the protein band of ␣ 1 * exhibited an apparent molecular mass of 53 kDa because of an additional glycosylated asparagine at posi- tion 80 of the newly introduced ␤ 3 subunit insert. The protein bands were quanti fi ed, and results obtained indicated that ␣ 1 * and ␣ subunits were expressed to a similar extent on the surface of transfected cells. Then, the Western blot was stripped and analyzed using digoxygenized ␤ 3 (1 – 13) antibodies (Fig. 4). Fi- nally, blots were again stripped and were probed with ␥ 2 (1 – 33) antibodies. Whereas similar amounts of ␤ 3 subunits (54 kDa) were coprecipitated with ␣ 1 subunits from ␣ 1 ␤ 3 ␥ 2 or ␣ 1 * ␤ 3 ␥ 2 transfected cells, the amount of ␥ 2 subunits (49 kDa) coprecipi- tated with ␣ 1 * subunits was only 32 Ϯ 3% (mean Ϯ SEM, n ϭ 3; from three different transfections) of that coprecipitated with ␣ 1 subunits. Similar results were obtained when the order of detec- tion of subunits was changed and Western blots were fi rst probed with ␥ 2 (1 – 33) antibodies and after stripping were re-analyzed with ␣ 1 (1 – 9) or ␤ 3 (1 – 13) antibodies. These results indicate that ␣ 1 * was able to form receptors with ␤ 3 subunits, but that the ability to form receptors containing ␥ 2 subunits was reduced by 68%. To investigate the properties of the receptors formed, HEK cells cotransfected with ␣ 1 *, ␤ 3 , and ␥ 2 subunits were subjected to patch-clamp analysis, and whole-cell recordings were compared with those from cells transfected with ␣ 1 , ␤ 3 , and ␥ 2 subunits. GABA exhibited an apparent EC 50 of 68 Ϯ 10 ␮ M (mean Ϯ SEM; n ϭ 11 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells transfected with ␣ 1 *, ␤ 3 , and ␥ 2 subunits and elicited a maximal current of 713 Ϯ 170 pA at a GABA concen- tration of 1000 ␮ M (Fig. 5 A ). In contrast, GABA exhibited an EC 50 of 7.7 Ϯ 2.3 ␮ M (mean Ϯ SEM; n ϭ 8 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells trans- fected with ␣ 1 , ␤ 3 , and ␥ 2 subunits and elicited a maximal current of 2988 Ϯ 469 pA at a concentration of 300 ␮ M (Fig. 5 B ). These data not only indicated that GABA exhibited a 10-fold reduced potency for activating ␣ * ␤ ␥ receptors, but also that the maxi- mal current of cells transfected with ␣ 1 * ␤ 3 ␥ 2 subunits was only ϳ 24% of that transfected with ␣ 1 ␤ 3 ␥ 2 subunits. Because surface expression studies indicated a signi fi cant for- mation of ␣ 1 * ␤ 3 receptors in ␣ 1 * ␤ 3 ␥ 2 transfected cells, the prop- erties of receptors in ␣ 1 * ␤ 3 transfected cells were also investi- gated. Although homo-oligomeric receptors composed of ␤ 3 subunits could also have been formed under the conditions used, they would not have contributed to the GABA evoked current because these receptors apparently are not gated by GABA (Connolly et al., 1996b). Using various GABA concentrations, it was demonstrated that GABA exhibited an EC 50 of 10.5 Ϯ 2.1 ␮ M (mean Ϯ SEM; n ϭ 8 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells transfected with ␣ 1 * and ␤ 3 subunits and elicited a maximal current of 270 Ϯ 63 pA at a concentration of 300 ␮ M (Fig. 5 C ). In contrast, GABA exhibited an EC 50 of 3.0 Ϯ 1.2 ␮ M (mean Ϯ SEM; n ϭ 9 cells from different plates; total of three transfections) (Fig. 5 E ) in cells transfected with ␣ 1 and ␤ 3 subunits and elicited a maximal current of 426 Ϯ 146 pA at a concentration of 100 ␮ M (Fig. 5 D ). These data supported the conclusion that the ␣ 1 * construct was able to form functional receptors with ␤ 3 subunits. The potency of GABA for activating ␣ 1 * ␤ 3 receptors, however, was signi fi cantly ( p Ͻ 0.05; Student ’ s t test) reduced compared with receptors composed of ␣ 1 ␤ 3 subunits. Similarly, the maximal currents elicited by GABA in ␣ 1 * ␤ 3 transfected cells were signi fi cantly smaller than those in ␣ 1 ␤ 3 transfected cells ( p Ͻ 0.05). Although ␣ 1 * ␤ 3 receptors signi fi cantly contribute to receptors formed in ␣ 1 * ␤ 3 ␥ 2 transfected HEK cells, as indicated by surface expression studies, because of the low maximum currents ob- served in ␣ 1 * ␤ 3 receptors (Fig. 5 C ), these receptors overall have a comparatively small contribution to currents elicited in ␣ 1 * ␤ 3 ␥ 2 transfected cells (Fig. 5 A ) that is apparent only as a slightly increased range of GABA concentrations that are able to elicit currents in these cells (Fig. 5 E ). Thus, most of the current elicited in the cells investigated was produced by ␣ 1 * ␤ 3 ␥ 2 receptors. The low apparent potency of GABA to activate currents in these cells as well as the increased dose range of GABA for stimulation of currents clearly indicated the formation of ␣ 1 * ␤ 3 ␥ 2 receptors in addition to ␣ 1 * ␤ 3 receptors. This conclusion was supported by investigating the effects of 100 ␮ M Zn 2 ϩ on whole-cell currents stimulated by ...

Context 2

... The precipitate was subjected to SDS-PAGE, and the proteins were detected with digoxygenized ␤ 3 (1 – 13) antibodies in Western blots. The actual expression of the chimeras was con- fi rmed by precipitation and detection with ␤ 3 (1 – 13) antibodies (data not shown). In ␤ 3 (1 – 115)chim ␣ 1 (101 – 117) the 17 C-terminal amino acids of the ␤ 3 (1 – 115) fragment were replaced by amino acids 101 – 117 of the ␣ subunit. As indicated in Figure 2, this chimera could not be coprecipitated with full-length ␥ 2 subunits from appropriately cotransfected HEK cells, demonstrating the speci fi city of the ␥ 2 (319 – 366) antibodies used and indicating that amino acids ␣ 1 (101 – 117) are not able to induce binding to ␥ 2 subunits. In ␤ 3 (1 – 115)chim ␣ 1 (80 – 117), the amino acid sequence ␤ 3 (78 – 115) was replaced by ␣ 1 (80 – 117). This construct was able to bind to full-length ␥ 2 subunits (Fig. 2), but not to full-length ␤ 3 subunits (data not shown). Because amino acids ␣ 1 (101 – 117) were not suf fi cient to induce binding to ␥ 2 subunits as discussed above, this indicated that amino acids 80 – 100 of the ␣ 1 subunit are important for binding to ␥ 2 subunits. To directly con fi rm this conclusion, the construct ␤ 3 (1 – 115)chim ␣ 1 (80 – 100) was generated (Fig. 2), in which amino acids ␣ 1 (80 – 100) were incorporated into ␤ 3 (1 – 115), replacing amino acids ␤ 3 (78 – 98). As expected, this chimera was able to bind to ␥ 2 subunits. To investigate which part of the ␣ 1 (80 – 100) sequence is re- sponsible for binding to ␥ 2 subunits, four additional chimeras were constructed. In ␤ 3 (1 – 115)chim ␣ 1 (80 – 86), amino acids ␤ 3 (78 – 84) were replaced by the amino acids ␣ 1 (80 – 86), in ␤ 3 (1 – 115)chim ␣ 1 (87 – 93) the sequence ␤ 3 (85 – 91) was replaced by ␣ 1 (87 – 93), in ␤ 3 (1 – 115)chim ␣ 1 (94 – 100) the sequence ␤ 3 (92 – 98) was replaced by amino acids ␣ 1 (94 – 100), and in ␤ 3 (1 – 115)chim ␣ 1 (80 – 93) the sequence ␤ 3 (78 – 91) was replaced by ␣ 1 (80 – 93) in the ␤ 3 (1 – 115) fragment. None of these chimeras was able to bind to ␥ 2 subunits. These results indicate that the whole ␣ (80 – 100) sequence is necessary for binding to ␥ subunits. To investigate the importance of the ␣ 1 (80 – 100) sequence not only for the assembly of truncated subunits and dimers, but also for assembly of full-length subunits and pentameric receptors, a full-length ␣ 1 chimera ( ␣ 1 *) was constructed in which the se- quence ␣ 1 (79 – 100) was replaced by the sequence ␤ 3 (77 – 98). The additional exchange of the amino acid 79 of the ␣ 1 subunit in ␣ 1 * was necessary to avoid the generation of two adjacent prolines that could have destroyed the conformation of the resulting chi- mera (Fig. 2). In control experiments, it was demonstrated that the extent of expression of the ␣ 1 * chimera was similar to that of the ␣ 1 subunit in HEK cells (data not shown). HEK cells were then cotransfected with ␣ 1*, ␤ 3 , and ␥ 2 sub- units and subunits expressed were investigated by immuno fl uo- rescence and confocal laser microscopy. As shown in Figure 3, ␣ 1 * (Fig. 3 A ) and ␤ 3 subunits (Fig. 3 B ) could be easily detected on the surface of intact cells, but for the ␥ 2 subunit only a weak labeling was observed (Fig. 3 C ), although the labeling of the ␥ 2 subunit in permeabilized cells (Fig. 3 F ) was comparable with that of ␣ 1 (Fig. 3 D ) and ␤ 3 (Fig. 3 E ) subunits. In HEK cells cotrans- fected with ␣ 1 , ␤ 3 , and ␥ 2 subunits, all three subunits could be detected on the cell surface (Fig. 3 G – I ). Because previous results have indicated that ␣ 1 subunits alone in contrast to ␣ 1 ␤ 3 subunit combinations do not form receptors that are incorporated into the plasma membrane to a signi fi cant extent, these results sug- gested that ␣ 1 * predominantly formed receptors with ␤ 3 subunits that are expressed on the cell surface, but the ability to form receptors containing ␥ 2 subunits was signi fi cantly reduced. To quantify this phenomenon, HEK cells were cotransfected with ␣ 1 , ␤ 3 , and ␥ 2 subunits or with ␣ 1 *, ␤ 3 , and ␥ 2 subunits. GABA A receptors expressed on the surface of the cells were labeled by an incubation of intact cells with ␣ 1 (1 – 9) antibodies. Antibody labeled receptors were extracted and precipitated by addition of Immunoprecipitin. The precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized ␣ 1 (1 – 9) antibodies (Fig. 4). In contrast to ␣ 1 subunits (51 kDa, the weak 46 kDa band presumably represents a degradation product), the protein band of ␣ 1 * exhibited an apparent molecular mass of 53 kDa because of an additional glycosylated asparagine at posi- tion 80 of the newly introduced ␤ 3 subunit insert. The protein bands were quanti fi ed, and results obtained indicated that ␣ 1 * and ␣ subunits were expressed to a similar extent on the surface of transfected cells. Then, the Western blot was stripped and analyzed using digoxygenized ␤ 3 (1 – 13) antibodies (Fig. 4). Fi- nally, blots were again stripped and were probed with ␥ 2 (1 – 33) antibodies. Whereas similar amounts of ␤ 3 subunits (54 kDa) were coprecipitated with ␣ 1 subunits from ␣ 1 ␤ 3 ␥ 2 or ␣ 1 * ␤ 3 ␥ 2 transfected cells, the amount of ␥ 2 subunits (49 kDa) coprecipi- tated with ␣ 1 * subunits was only 32 Ϯ 3% (mean Ϯ SEM, n ϭ 3; from three different transfections) of that coprecipitated with ␣ 1 subunits. Similar results were obtained when the order of detec- tion of subunits was changed and Western blots were fi rst probed with ␥ 2 (1 – 33) antibodies and after stripping were re-analyzed with ␣ 1 (1 – 9) or ␤ 3 (1 – 13) antibodies. These results indicate that ␣ 1 * was able to form receptors with ␤ 3 subunits, but that the ability to form receptors containing ␥ 2 subunits was reduced by 68%. To investigate the properties of the receptors formed, HEK cells cotransfected with ␣ 1 *, ␤ 3 , and ␥ 2 subunits were subjected to patch-clamp analysis, and whole-cell recordings were compared with those from cells transfected with ␣ 1 , ␤ 3 , and ␥ 2 subunits. GABA exhibited an apparent EC 50 of 68 Ϯ 10 ␮ M (mean Ϯ SEM; n ϭ 11 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells transfected with ␣ 1 *, ␤ 3 , and ␥ 2 subunits and elicited a maximal current of 713 Ϯ 170 pA at a GABA concen- tration of 1000 ␮ M (Fig. 5 A ). In contrast, GABA exhibited an EC 50 of 7.7 Ϯ 2.3 ␮ M (mean Ϯ SEM; n ϭ 8 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells trans- fected with ␣ 1 , ␤ 3 , and ␥ 2 subunits and elicited a maximal current of 2988 Ϯ 469 pA at a concentration of 300 ␮ M (Fig. 5 B ). These data not only indicated that GABA exhibited a 10-fold reduced potency for activating ␣ * ␤ ␥ receptors, but also that the maxi- mal current of cells transfected with ␣ 1 * ␤ 3 ␥ 2 subunits was only ϳ 24% of that transfected with ␣ 1 ␤ 3 ␥ 2 subunits. Because surface expression studies indicated a signi fi cant for- mation of ␣ 1 * ␤ 3 receptors in ␣ 1 * ␤ 3 ␥ 2 transfected cells, the prop- erties of receptors in ␣ 1 * ␤ 3 transfected cells were also investi- gated. Although homo-oligomeric receptors composed of ␤ 3 subunits could also have been formed under the conditions used, they would not have contributed to the GABA evoked current because these receptors apparently are not gated by GABA (Connolly et al., 1996b). Using various GABA concentrations, it was demonstrated that GABA exhibited an EC 50 of 10.5 Ϯ 2.1 ␮ M (mean Ϯ SEM; n ϭ 8 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells transfected with ␣ 1 * and ␤ 3 subunits and elicited a maximal current of 270 Ϯ 63 pA at a concentration of 300 ␮ M (Fig. 5 C ). In contrast, GABA exhibited an EC 50 of 3.0 Ϯ 1.2 ␮ M (mean Ϯ SEM; n ϭ 9 cells from different plates; total of three transfections) (Fig. 5 E ) in cells transfected with ␣ 1 and ␤ 3 subunits and elicited a maximal current of 426 Ϯ 146 pA at a concentration of 100 ␮ M (Fig. 5 D ). These data supported the conclusion that the ␣ 1 * construct was able to form functional receptors with ␤ 3 subunits. The potency of GABA for activating ␣ 1 * ␤ 3 receptors, however, was signi fi cantly ( p Ͻ 0.05; Student ’ s t test) reduced compared with receptors composed of ␣ 1 ␤ 3 subunits. Similarly, the maximal currents elicited by GABA in ␣ 1 * ␤ 3 transfected cells were signi fi cantly smaller than those in ␣ 1 ␤ 3 transfected cells ( p Ͻ 0.05). Although ␣ 1 * ␤ 3 receptors signi fi cantly contribute to receptors formed in ␣ 1 * ␤ 3 ␥ 2 transfected HEK cells, as indicated by surface expression studies, because of the low maximum currents ob- served in ␣ 1 * ␤ 3 receptors (Fig. 5 C ), these receptors overall have a comparatively small contribution to currents elicited in ␣ 1 * ␤ 3 ␥ 2 transfected cells (Fig. 5 A ) that is apparent only as a slightly increased range of GABA concentrations that are able to elicit currents in these cells (Fig. 5 E ). Thus, most of the current elicited in the cells investigated was produced by ␣ 1 * ␤ 3 ␥ 2 receptors. The low apparent potency of GABA to activate currents in these cells as well as the increased dose range of GABA for stimulation of currents clearly indicated the formation of ␣ 1 * ␤ 3 ␥ 2 receptors in addition to ␣ 1 * ␤ 3 receptors. This conclusion was supported by investigating the effects of 100 ␮ M Zn 2 ϩ on whole-cell currents stimulated by 100 ␮ M GABA. In agreement with previous results (Draguhn et al., 1990; Gingrich and Burkat, 1998), currents mediated by the wild-type ␣ 1 ␤ 3 ␥ 2 receptors were only weakly reduced (86 Ϯ 4% of control; n ϭ 6; total of four transfections), whereas currents mediated by ␣ 1 ␤ 3 receptor were reduced to 7 Ϯ 3% ( n ϭ 6; total of three transfections) (Fig. 5 F ) in the presence of Zn 2 ϩ . For HEK cells transfected with ␣ 1 *, ␤ 3 , and ␥ 2 subunits, currents mediated by 100 ␮ M GABA were reduced to 50 Ϯ 4% ( n ϭ 7; total of four transfections), and for ...

Context 3

... total of four transfections) (Fig. 5 E ) in HEK cells transfected with ␣ 1 *, ␤ 3 , and ␥ 2 subunits and elicited a maximal current of 713 Ϯ 170 pA at a GABA concen- tration of 1000 ␮ M (Fig. 5 A ). In contrast, GABA exhibited an EC 50 of 7.7 Ϯ 2.3 ␮ M (mean Ϯ SEM; n ϭ 8 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells trans- fected with ␣ 1 , ␤ 3 , and ␥ 2 subunits and elicited a maximal current of 2988 Ϯ 469 pA at a concentration of 300 ␮ M (Fig. 5 B ). These data not only indicated that GABA exhibited a 10-fold reduced potency for activating ␣ * ␤ ␥ receptors, but also that the maxi- mal current of cells transfected with ␣ 1 * ␤ 3 ␥ 2 subunits was only ϳ 24% of that transfected with ␣ 1 ␤ 3 ␥ 2 subunits. Because surface expression studies indicated a signi fi cant for- mation of ␣ 1 * ␤ 3 receptors in ␣ 1 * ␤ 3 ␥ 2 transfected cells, the prop- erties of receptors in ␣ 1 * ␤ 3 transfected cells were also investi- gated. Although homo-oligomeric receptors composed of ␤ 3 subunits could also have been formed under the conditions used, they would not have contributed to the GABA evoked current because these receptors apparently are not gated by GABA (Connolly et al., 1996b). Using various GABA concentrations, it was demonstrated that GABA exhibited an EC 50 of 10.5 Ϯ 2.1 ␮ M (mean Ϯ SEM; n ϭ 8 cells from different plates; total of four transfections) (Fig. 5 E ) in HEK cells transfected with ␣ 1 * and ␤ 3 subunits and elicited a maximal current of 270 Ϯ 63 pA at a concentration of 300 ␮ M (Fig. 5 C ). In contrast, GABA exhibited an EC 50 of 3.0 Ϯ 1.2 ␮ M (mean Ϯ SEM; n ϭ 9 cells from different plates; total of three transfections) (Fig. 5 E ) in cells transfected with ␣ 1 and ␤ 3 subunits and elicited a maximal current of 426 Ϯ 146 pA at a concentration of 100 ␮ M (Fig. 5 D ). These data supported the conclusion that the ␣ 1 * construct was able to form functional receptors with ␤ 3 subunits. The potency of GABA for activating ␣ 1 * ␤ 3 receptors, however, was signi fi cantly ( p Ͻ 0.05; Student ’ s t test) reduced compared with receptors composed of ␣ 1 ␤ 3 subunits. Similarly, the maximal currents elicited by GABA in ␣ 1 * ␤ 3 transfected cells were signi fi cantly smaller than those in ␣ 1 ␤ 3 transfected cells ( p Ͻ 0.05). Although ␣ 1 * ␤ 3 receptors signi fi cantly contribute to receptors formed in ␣ 1 * ␤ 3 ␥ 2 transfected HEK cells, as indicated by surface expression studies, because of the low maximum currents ob- served in ␣ 1 * ␤ 3 receptors (Fig. 5 C ), these receptors overall have a comparatively small contribution to currents elicited in ␣ 1 * ␤ 3 ␥ 2 transfected cells (Fig. 5 A ) that is apparent only as a slightly increased range of GABA concentrations that are able to elicit currents in these cells (Fig. 5 E ). Thus, most of the current elicited in the cells investigated was produced by ␣ 1 * ␤ 3 ␥ 2 receptors. The low apparent potency of GABA to activate currents in these cells as well as the increased dose range of GABA for stimulation of currents clearly indicated the formation of ␣ 1 * ␤ 3 ␥ 2 receptors in addition to ␣ 1 * ␤ 3 receptors. This conclusion was supported by investigating the effects of 100 ␮ M Zn 2 ϩ on whole-cell currents stimulated by 100 ␮ M GABA. In agreement with previous results (Draguhn et al., 1990; Gingrich and Burkat, 1998), currents mediated by the wild-type ␣ 1 ␤ 3 ␥ 2 receptors were only weakly reduced (86 Ϯ 4% of control; n ϭ 6; total of four transfections), whereas currents mediated by ␣ 1 ␤ 3 receptor were reduced to 7 Ϯ 3% ( n ϭ 6; total of three transfections) (Fig. 5 F ) in the presence of Zn 2 ϩ . For HEK cells transfected with ␣ 1 *, ␤ 3 , and ␥ 2 subunits, currents mediated by 100 ␮ M GABA were reduced to 50 Ϯ 4% ( n ϭ 7; total of four transfections), and for cells transfected with ␣ 1 * and ␤ 3 subunits, GABA-mediated currents were reduced to 8 Ϯ 2% ( n ϭ 8; total of four transfections) in the presence of 100 ␮ M Zn 2 ϩ (Fig. 5 F ). Because ␣ 1 ␤ 3 and ␣ 1 * ␤ 3 receptors exhibit a comparable Zn 2 ϩ sensitivity, it is reasonable to assume that the Zn 2 ϩ sensitivity of ␣ 1 * ␤ 3 ␥ 2 and ␣ 1 ␤ 3 ␥ 2 receptors was also comparable. The 36% increase in Zn 2 ϩ sensitivity of ␣ 1 * ␤ 3 ␥ 2 -transfected cells therefore indicated that ϳ 40% of the ␣ 1 * ␤ 3 ␥ 2 current was mediated by the additionally formed ␣ 1 * ␤ 3 receptors. Combined with the observation that the main conductance level of ␣␤ receptors (15 – 18 pS) is only half of that of ␣␤␥ receptors ( ϳ 30 pS; Hevers and L ̈ ddens, 1998), and assuming that the same holds true for ␣ 1 * ␤ 3 and ␣ 1 * ␤ 3 ␥ 2 receptors, a ratio of ␣ 1 * ␤ 3 : ␣ 1 * ␤ 3 ␥ 2 receptors of 80:60 can be calculated, indicating that ␣ 1 * ␤ 3 ␥ 2 receptors represented 43% of receptors formed in these cells. Given the many assumptions that had to be made in the course of this calculation, this percentage is in good agreement with the data from the immunoprecipitation experiments shown in Figure 4. Recently it has been demonstrated that incorporation of the amino acid sequence ␥ (91 – 104) into the fragment ␣ (1 – 100), that per se could not bind to ␣ 1 subunits, resulted in the chimera ␣ 1 (1 – 100)chim ␥ 2 (91 – 104) that was able to bind to ␣ 1 subunits. From this it was concluded that the amino acid sequence ␥ 2 (91 – 104) forms the contact site to ␣ 1 subunits (Klausberger et al., 2000). It therefore seemed interesting to investigate whether the ␥ 2 (91 – 104) sequence directly interacts with the ␣ 1 (80 – 100) sequence. To clarify this question, it fi rst was investigated whether the ␣ 1 (1 – 100) fragment and the fragment ␤ 3 (1 – 115), which was used to identify the ␣ 1 (80 – 100) contact site (Fig. 2), could bind to each other. For this, fragments ␤ 3 (1 – 115) and ␣ 1 (1 – 100) were cotrans- fected into HEK cells. The extract of HEK cells expressing ␤ 3 (1 – 115) and ␣ 1 (1 – 100) fragments was then immunoprecipitated with ␤ 3 (1 – 13) antibodies, and the precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized ␣ 1 (1 – 9) antibodies. As shown in Figure 6, A and B , ␣ 1 (1 – 100) fragments were not coprecipitated by ␤ 3 (1 – 13) antibodies, con- fi rming the absence of cross-reactivity of these antibodies with the ␣ 1 (1 – 100) fragments and indicating that ␤ 3 (1 – 115) could not bind to ␣ 1 (1 – 100) fragments. Similarly, the construct ␤ 3 (1 – 115)chim ␣ 1 (80 – 100), which contains the putative binding site for ␥ 2 subunits, was unable to bind to ␣ 1 (1 – 100) fragments after cotransfection into HEK cells (Fig. 6 A,B ). In the reverse exper- iment, the construct ␣ 1 (1 – 100)chim ␥ 2 (91 – 104), containing the binding site for ␣ 1 subunits, could also not bind to the ␤ 3 (1 – 115) fragment. Only when the ␣ 1 (80 – 100) sequence was incorporated into the ␤ 3 (1 – 115) fragment and the ␥ 2 (91 – 104) sequence was incorporated into the ␣ 1 (1 – 100) fragment, the resulting chimeras could bind to each other (Fig. 6 A,B ). These results indicate that the ␣ 1 (80 – 100) and the ␥ 2 (91 – 104) sequences can directly bind to each other. In control experiments (Fig. 6 C ) it was demonstrated that each of the constructs used in this experiment was expressed to a similar extent after single transfection into HEK cells. Constructs ␣ 1 (1 – 100) and ␣ 1 (1 – 100)chim ␥ 2 (91 – 104) each contained a single glycosylation site and thus, gave rise to two fragments: a weakly labeled of 12 kDa and a strongly labeled of 14 kDa. Construct ␤ 3 (1 – 115) contained two glycosylation sites and thus, formed three fragments, two strongly labeled of 16 and 18 kDa and a weakly labeled fragment of 14 kDa. Construct ␤ 3 (1 – 115)chim ␣ 1 (80 – 100) contained only one glycosylation site and formed a strongly labeled protein band of 14 and weakly labeled band of 16 kDa. Interestingly, predominantly the unglycosylated ␣ 1 (1 – 100)chim ␥ 2 (91 – 104) fragment of 12 kDa seemed to assemble with ␤ 3 (1 – 115)chim ␣ 1 (80 – 100) on cotransfection of these fragments into HEK cells, although the glycosylated fragment of 14 kDa was the predominant one expressed after single transfection of HEK cells (Figs. 1 B, 6 C ). This suggests that assembly of subunit frag- ments already starts when subunits are not fully glycosylated. This conclusion is supported by previous observations (Klausberger et al., 2000, 2001) as well as by observations with other constructs (Fig. 1 B ). The present study demonstrated that the N-terminal extracellular domain of the ␣ 1 subunit [ ␣ 1 (1 – 221)] could bind to full-length ␥ 2 subunits after coexpression in HEK cells, as indicated by coim- munoprecipitation with subunit-speci fi c antibodies. Binding be- tween ␣ (1 – 221) and ␥ subunits represented a speci fi c assembly process, as indicated by the formation of speci fi c [ 3 H]Ro15 – 1788 binding sites that are assumed to be formed on the interface of ␣ 1 and ␥ 2 subunits of GABA A receptors. These results are consistent with previous studies indicating that N-terminal sequences of GABA A receptor (Hackam et al., 1997; Klausberger et al., 2000) or K ϩ channel (Shen et al., 1993) subunits can assemble with full-length subunits. A subsequent reduction in the size of the truncated subunit indicated that the ␣ 1 (1 – 117), but not the ␣ 1 (1 – 100) construct was still able to bind to ␥ 2 subunits. The respective binding site was then identi fi ed by incorporating various ␣ 1 sequences into the ␤ 3 (1 – 115) fragment. This fragment is homologous to ␣ 1 (1 – 117) but in contrast to the latter construct could not bind to ␥ 2 subunits after coexpression in HEK cells. The incorporation of the se- quence ␣ 1 (80 – 100) into the ␤ 3 (1 – 115) fragment was suf fi cient to induce binding to ␥ 2 but not to ␤ 3 subunits, suggesting that the ␣ 1 binding sites for ␥ 2 and ␤ 3 subunits are different. The observation that the ␣ 1 (1 – 100) fragment was unable to bind to ␥ 2 subunits although it contained the ␣ 1 (80 – 100) se- ...

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... *, 3 , and 2 subunits. GABA A receptors expressed on the surface of the cells were labeled by an incubation of intact cells with 1 (1-9) antibodies. Antibody labeled receptors were extracted and precipitated by addition of Immunoprecipitin. The precipitate was subjected to SDS-PAGE and Western blot analysis using digoxygenized 1 (1-9) antibodies (Fig. 4). In contrast to 1 subunits (51 kDa, the weak 46 kDa band presumably represents a degradation product), the protein band of 1 * exhibited an apparent molecular mass of 53 kDa because of an additional glycosylated asparagine at posi- tion 80 of the newly introduced 3 subunit insert. The protein bands were quantified, and results obtained ...

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... an additional glycosylated asparagine at posi- tion 80 of the newly introduced 3 subunit insert. The protein bands were quantified, and results obtained indicated that 1 * and 1 subunits were expressed to a similar extent on the surface of transfected cells. Then, the Western blot was stripped and analyzed using digoxygenized 3 (1-13) antibodies (Fig. 4). Fi- nally, blots were again stripped and were probed with 2 (1-33) antibodies. Whereas similar amounts of 3 subunits (54 kDa) were coprecipitated with 1 subunits from 1 3 2 or 1 * 3 2 transfected cells, the amount of 2 subunits (49 kDa) coprecipi- tated with 1 * subunits was only 32 3% (mean SEM, n 3; from three different ...

Context 6

... with the observation that the main conductance level of receptors (15-18 pS) is only half of that of receptors (30 pS;Hevers and Lüddens, 1998), and assuming that the same holds true for 1 * 3 and 1 * 3 2 receptors, a ratio of 1 * 3 : 1 * 3 2 receptors of 80:60 can be calculated, indicating that 1 * 3 2 receptors represented 43% of receptors formed in these cells. Given the many assumptions that had to be made in the course of this calculation, this percentage is in good agreement with the data from the immunoprecipitation experiments shown in Figure 4. . Western blot analysis of GABA A receptors labeled on the surface of HEK cells. ...