Protein movements underlying ligand-gated ion channel activation are poorly understood. Here we used disulfide bond trapping to examine the proximity and mobility of cysteines substituted for aligned GABAA receptor alpha1 and beta1 M2 segment channel-lining residues in resting and activated receptors. With or without GABA, disulfide bonds formed at alpha1N275C/beta1E270C (20') and alpha1S272C/beta1H267C (17'), near the extracellular end, suggesting that this end is more mobile and/or flexible than the rest of the segment. Near the middle of M2, at alpha1T261C/beta1T256C (6'), a disulfide bond formed only in the presence of GABA and locked the channels open. Channel activation must involve an asymmetric rotation of two adjacent subunits toward each other. This would move aligned engineered cysteines on different subunits into proximity and allow disulfide bond formation without blocking conduction. Asymmetric rotation of M2 segments is probably a common gating mechanism in other ligand-gated ion channels.

The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showing that CAP sequesters monomeric actin (Kd in the range of 0.5-5 microM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.

The ␥-aminobutyric acid, type A (GABA A ), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn 2؉ -binding sites. Disulfide bonds formed in ␣ 1 ␤ 1 E270C␥ 2 but not in ␣ 1 N275C␤ 1 ␥ 2 or ␣ 1 ␤ 1 ␥ 2 K285C. Diazepam potentiation and Zn 2؉ inhibition demonstrated that expressed receptors contained a ␥ subunit. Therefore, the disulfide bond in ␣ 1 ␤ 1 E270C␥ 2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-Å resolution structure, the distance between ␣ carbon atoms of 20 aligned positions in non-adjacent subunits is ϳ19 Å. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn 2؉ -binding sites whose affinity depends on the energetics of forming a bidentatebinding site. The Zn 2؉ inhibition IC 50 for ␣ 1 ␤ 1 E270C␥ 2 was 34 nM. In contrast, it was greater than 100 M in ␣ 1 N275C␤ 1 ␥ 2 and ␣ 1 ␤ 1 ␥ 2 K285C receptors. The high Zn 2؉ affinity in ␣ 1 ␤ 1 E270C␥ 2 implies that this region in the ␤ subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the ␤ and ␣ M2 segments may have important implications for GABA-induced conformational changes during channel gating.GABA A 1 receptors are allosteric proteins that mediate fast inhibitory neurotransmission in the central nervous system (1-3). They are members of the Cys-loop receptor ion channel gene superfamily that includes glycine, serotonin type 3 (5-HT 3 ), and nicotinic acetylcholine (ACh) receptors (4 -6). GABA A receptors are formed by five homologous subunits assembled around a central channel. Most endogenous receptors contain two ␣, two ␤, and one ␥ subunit arranged in a clockwise orientation ␣␤␣␤␥ when observed from the extracellular end of the channel (7,8). However, expression of just ␣ and ␤ subunits also results in functional receptors with the favored stoichiometry being two ␣ and three ␤ in the order ␣␤␣␤␤ (9 -11). Each subunit has an ϳ200-amino acid, extracellular, N-terminal, ligand-binding domain and a C-terminal, channel-forming domain with four membrane-spanning segments (M1, M2, M3, and M4).The channel is principally lined by the five ␣-helical M2 segments (12, 13). An index numbering system facilitates comparisons between M2 segments of superfamily members (14). The 0Ј position is defined as the positively charged residue located near the cytoplasmic end of the channel, GABA A ␤ 1 R250. The 20Ј position, GABA A ␤ 1 E270, is aligned with the acetylcholine receptor extracellular ring of charge (15) and is predi...