Effect of membrane voltage on off-response currents. (A–E) Representative current traces recorded for the indicated chimeras. ACh, 300 μM; externally applied Zn²⁺, 1 mM; wash, physiological solution (ND96). Color code for holding voltages in mV is as follows: magenta, -100; green, -120; brown, -140; blue, -160; red, -180; black, -200. The gray traces correspond to steady-state activation currents elicited by ACh at -200 (A and B), -180 (C and D), and -160 (E) mV. (F) Linear regression of the ratio of off-response peak currents over the OB population plotted as a function of membrane voltage. OB is calculated by subtracting the current observed at steady-state inhibition from the current of steady-state activation (i.e., the current lost due to the block). Error bars correspond to SD of at least five independent experiments.

Effects of preceding application of Zn²⁺ on activation time courses. (A–H) External application (6 s) of 1 mM Zn²⁺ is followed by application of 300 μM ACh alone (black traces) to oocytes expressing the depicted chimeras. The gray traces correspond to activation by ACh without preceding application of Zn²⁺. Time constants of activation are provided in Table 2.

Effects of Zn²⁺ applied externally after steady-state activation by ACh is achieved (Zn²⁺ postapplication). (A, B, D, and E) Representative current traces of chimeras containing histidines (A and B) or glycines (D and E)atthe indicated positions. ACh, 300 μM (saturating concentration); Zn²⁺, increasing μM concentrations as depicted. (C) Dose–response isotherms expressing fractional responses as a function of Zn²⁺ concentrations. Open circles, chimera H-5′; filled circles, chimera H-2′. (F) Inhibition isotherms corresponding to the control mutants. Open circles, G-5′; filled circles, G-2′. Data were fitted with a nonlinear regression to the Hill equation (Supporting Materials and Methods in Supporting Text). All recordings were performed at -80-mV holding potential.

Effects of Zn²⁺ applied externally before activation of chimera H-2′. (A) Application of Zn²⁺ (1 mM) for 0.5 (red), 2 (blue), and 6 (green) s is followed by coapplication of ACh (300 μM) together with Zn²⁺ (1 mM). Black trace corresponds to coapplication without preceding application of Zn²⁺. Dashed line corresponds to a wash with ND 96. (Inset) The ratio of off-response peak current over steady-state (SS) activation (ACh alone, gray trace) in relation to the time of Zn²⁺ application before the coapplication (open bars). The gray bar (Post) corresponds to the same ratio obtained in Zn²⁺ postapplication experiments (e.g., Fig. 2B, blue trace). (B) Application of Zn²⁺ (1 mM) for 6 s is followed by varying periods of coapplication of ACh (300 μM) plus Zn²⁺ (1 mM): purple, 0.25 s; red, 0.5 s; brown, 1 s; blue, 2 s; black, 6 s. In Inset, data were fitted with a nonlinear regression to Eq. 3 in Supporting Materials and Methods in Supporting Text. (C) Preapplication of Zn²⁺ for 2, 10, and 180 s is immediately followed by washout. All currents were recorded at -160 mV. Traces shown in A and C are from the same oocyte; the traces in B are from another oocyte, demonstrating the reproducibility of the currents' proportionalities. Experiments shown in A and B were repeated independently seven times; the error bars in Insets correspond to their SD values. Results shown in C were repeated independently five times.

Conformations and gating mechanism of a Cys-loop receptor pore. (A) Molecular surface of the membrane-embedded domain of chimera H-5′ displaying closed (Left) or open (Right) pore conformations, as viewed from within the membrane. For better viewing, the two frontal subunits have been removed; the carbons of the rear and frontal subunits are colored yellow and white, respectively. In all three subunits, oxygen, nitrogen, sulfur, and hydrogen atoms are colored red, blue, orange, and white, respectively. The black horizontal lines delineate the putative location of the membrane. Image was prepared with pymol. (B) Top view of the closed (Left) vs. open (Right) constriction as seen from the extracellular milieu. The residues from position -5′ to position 2′ are shown as space-filling spheres; carbon, nitrogen, oxygen, and hydrogen atoms are colored white, blue, red, and white, respectively. Note that the side chain of the conserved basic amino acid at position 0′ points outward from the permeation pathway (see also Note 3 in Supporting Text). The helical transmembrane segments (four per each of five differently colored subunits) are shown as cylinders. Image was prepared with pymol. (C) 2D schematic side view corresponding to the proposed gating motions, as shown for two facing subunits. Red and black lines represent the closed and open states, respectively. The plausible axis of tilting (shown as a gray ball) is perpendicular to the viewer and is located between positions 2′ and 9′. The gray arrows indicate the motions around this point, which remains fixed during tilting.