Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A  shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B  shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars ) and 5βTHDOC (blue bars ), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars  show data for receptors with both steroid-binding sites intact; the top pair  shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs  show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar  gives the probability that the potentiation differs from 1 (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001) and the number of oocytes studied. For additional information, see the full publication.41C  shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D  summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars  summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair  is for combinations with all sites intact or with both steroid sites mutated. The middle group of three  shows combinations for which the steroid site in the “a” pair is intact, while the upper group  shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B . For additional information, see the full publication.47GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.

Fig. 3. Studies of neurosteroid potentiation of GABAAreceptors. A  shows an image of the receptors assembled by the concatemers used for the first set of studies. For reference, the transmitter-binding pairs are identified by a subscript; for example, the binding pair flanked by α and γ subunits is designated “a.”B  shows data using two structurally distinct neurosteroids, 5αTHDOC (red bars ) and 5βTHDOC (blue bars ), on receptors that have specific steroid-binding sites ablated (both GABA-binding sites were intact in these constructs). The location and status of steroid-binding sites are summarized in the label on the left; for example, a(S)-b(S) means that both the αaand αbsubunits have wild-type Q241 residues, while a(−)/b(−) indicates that αahas the mutated Q241L residue. The bottom pair of bars  show data for receptors with both steroid-binding sites intact; the top pair  shows the loss of potentiation when both steroid-binding sites are mutated; and the middle pairs  show responses when a single site is selectively removed. Note that 1 μM 5αTHDOC potentiates more for all of the combinations with at least one intact site, indicating similar pharmacological properties. Potentiation of the a(S)-b(−) combination did not differ from combination with two intact steroid sites, but potentiation for the a(−)-b(S) combination was significantly less (ANOVA with Bonferroni correction). The data show mean +SE for potentiation by 1 μM neurosteroid. The legend to the right of each bar  gives the probability that the potentiation differs from 1 (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001) and the number of oocytes studied. For additional information, see the full publication.41,C  shows an image of the receptors assembled by the concatemers used for the studies of interactions between steroid-binding and transmitter-binding sites. D  summarizes the results for potentiation by 1 μM allopregnanolone. The legend to the left of the bars  summarizes the constructs used in terms of the GABA- and steroid-binding sites. For example, the legend a(G−)-b(−S) indicates that for the “a” binding pair, the GABA site is intact (βY205) and the steroid site is not (αQ241L) while for the “b” binding pair the GABA site is ablated (βY205S) and the steroid site is intact (αQ241). The bottom pair  is for combinations with all sites intact or with both steroid sites mutated. The middle group of three  shows combinations for which the steroid site in the “a” pair is intact, while the upper group  shows combinations for which the steroid site in the “b” pair is intact. For these combinations, the only difference which was significant was for the a(G−)-b(G−) comparison to a(GS)-b(GS). The legends are as in B . For additional information, see the full publication.47GABA = γ-aminobutyric acid; GABAA = γ-aminobutyric acid type A.

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