McLaughlin JT , Barron SC , See JA , Rosenberg RL .

DA017882 NIDA NIH HHS , R01 DA017882 NIDA NIH HHS

	Figure 1. A model of the α7 AChR extracellular domain. Ribbon cartoon showing two of the five subunits viewed from the outside. In the subunit to the left of the central interface (yellow), the outer β sheet in is highlighted in orange, the transition zone E44 residue is orange, and the W148 residue is shown in gray to identify the ACh binding pocket (Zhong et. al., 1998). The subunit to the right shows a view of the inner sheet (teal), and other residues targeted in this study. The sequence surround mutants characterized in this study is shown beneath the cartoon: M37, M40 cyan; N52 green; N170 blue; E172 purple.
	Figure 2. Positive allosteric modulation by divalent cations requires E44. ACh dose-response curves for the parental C115A/L247T (A) and the E44C mutant (B) in the absence (open squares) and presence (filled squares) of 10 mM BaCl2. Data are fitted to the Hill equation (solid lines). The positive allosteric modulation (leftward shift in the dose response curve) typically exhibited by α7 AChRs (A) is lost in the E44C mutant (B). Data are mean values (± SEM) from three determinations, normalized to the maximal value of the Hill equation fit of each data set. Hill coefficients for C115A/L247T (A): 2.5 ± 0.2 (open squares, -Ba2+), 1.9 ± 0.4 (filled squares, +Ba2+); and for the E44C mutant (B) 1.7 ± 0.2 (open squares, -Ba2+), 2.9 ± 0.7 (filled squares, +Ba2+).
	Figure 3. Barium slows the rate of MTSEA modification at M37C. Example of experimental paradigm used to assess Ba2+ effects on modification rates. (A) Successive ACh-evoked current traces recorded before and after repeated exposures to MTSEA (5 μM, 15 seconds), showing a decrement in responses to 30 μM ACh. Endpoints of MTSEA modification are determined by prolonged application of 500 μM MTSEA (right). (B) The same protocol, including Ba2+ pretreatment and co-application with MTSEA. Current traces are truncated in both (A) and (B) between consecutive MTSEA applications; in all cases the currents were allowed to return to baseline prior to the next application of MTSEA ± ACh. (C) Peak current amplitudes from (A) and (B) are normalized and plotted versus total MTSEA exposure time. Data from this single experiment (no error bars) are fitted to a single-exponential decay (solid line) to extract an apparent pseudo first-order rate constant. The pseudo first-order rate constants calculated in this experiment were 0.011 s-1 and 0.0019 s-1 for control (A) and +Ba2+ (B) measurements, respectively. Second-order rate constants are calculated from these values (Figures 4–6; Table 2).
	Figure 4. Barium alters the rate of MTSEA modification at inner β sheet residues. (A) Using the protocol described in Figure 3, we determined second-order rate constants for three reporter residues in the α7 AChR inner β sheet (M37C, M40C, and N52C). Mean values for second-order rate constants for modification by MTSEA alone (control), MTSEA + ACh, and MTSEA + Ba2+ are shown. * Rate was significantly different from control (P < 0.05). (B) A plot of the ratios of second-order rate constants. Ba2+ and ACh both slowed the rates of modification of M37C and M40C. At N52C, however, the rate of modification in the presence of Ba2+ was not significantly different from control, while ACh accelerated the modification rate. See Table 2 for summary including (n) for each condition.
	Figure 5. Barium alters the rate of MTSEA modification at residues required for modulation by divalent cations. Second-order rate constants were measured for three residues in the "transition zone" of the α7 AChR (E44C, N170C, and E172C). (A) Mean values for second-order rate constants for modification by MTSEA alone, MTSEA + ACh, and MTSEA + Ba2+. Ba2+ caused a significant decrease in MTSEA modification rates of both E44C and E172C, despite the loss of divalent cation-dependent modulation. Ba2+ did not have a significant effect on the modification rate of N170C, although ACh significantly increased the rate of modification of this residue. *Rate was significantly different from control (P < 0.05). ‡Rate was significantly different from that obtained in presence of ACh (P < 0.05). The plot of rate constant ratios (B) shows that the effect of Ba2+ on the rate of modification of E44C was significantly less than the effect of ACh. See Table 2 for summary including (n) for each condition.
	Figure 6. Charge neutralization at E172 does not alter the rate of modification M40C by MTSEA. Mean values for second-order modification rate constants for M40C (left, data from Fig. 3) compared to those obtained in receptors containing the E172Q mutation (M40C/E172Q). *Rates were significantly different from control (P < 0.05). See Table 2 for summary, including (n) for each condition.

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