Skip to main content
. 2015 Jul 30;290(38):23432–23446. doi: 10.1074/jbc.M115.672006

FIGURE 8.

FIGURE 8.

In α1β3γ2 GABAARs, GABA enhances S-[3H]mTFD-MPPB photolabeling at the β+ interface (β3Phe-289 in M3 and β3Thr-262 in M2) and the β− interface (β3Met-227 and β3Leu-231 in M1). 3H cpm (●, ○) and PTH-derivatives (□) released during N-terminal sequencing of fragments before βM3 (A), βM1 (B), and βM2 (C) from GABAARs photolabeled in the presence of bicuculline (●, 30 μm) or GABA (○, 300 μm). From the photolabeling experiment of Fig. 5, A and B, EndoLys-C digests of the 59-kDa gel bands were fractionated by rpHPLC, and materials in fractions 26 and 27 (A) or fractions 28–30 (B) were sequenced. A, primary sequence began at β3Ala-280 (I0 = 1.6 pmol), with a secondary sequence beginning at β3Arg-216 (I0 = ∼1 pmol). The major peak of 3H release at cycle 10, consistent with photolabeling of β3Phe-289, was enhanced by 100% in the presence of GABA (Table 1). B, primary sequence began at β3Arg-216 (I0 = 4.5 pmol), with a secondary sequence beginning at β3Ala-280, present at levels below 1 pmol before OPA treatment in cycle 13 and undetectable after treatment. The peaks of 3H release in cycles 12 and 16 indicated GABA-enhanced photolabeling of β3Met-227 and β3Leu-231 in βM1. The peak of 3H release in cycle 10 corresponds to photolabeling of β3Phe-289 in βM3 of the secondary sequence. C, to identify photolabeling in βM2, aliquots from the 61-kDa gel bands from GABAARs photolabeled in presence of GABA (○) or bicuculline (●) were sequenced after treatment of the sequencing filter with BNPS-skatole to cleave at the C termini of tryptophans. The sequence beginning at β3Ile-242 was present (I0 = 2.3 pmol), along with fragments at 1–4 pmol each beginning at the β3 subunit N terminus, β3Arg-68, β3Val-93, and β3Arg-169, 49 amino acids before βM1, β3Ser-427, and β3Leu-444 in βM4 that are 17 and 4 amino acids in length, respectively. The peak of 3H release in cycle 21 indicated GABA-enhanced photolabeling of β3Thr-262 (βM2–12′). No 3H release was seen when intact β subunit was sequenced, and if cycle 21 of the β3Arg-169 fragment had been photolabeled, it would have been recovered by rpHPLC from EndoLys-C digests as a hydrophilic fragment from the ECD.