Skip to main content
. 2010 Oct 7;1(12):796–809. doi: 10.1021/cn100073x

Figure 5.

Figure 5

Effect of MTSEA on ACh-induced currents. Currents recorded from oocytes expressing either wildtype α4, α4T2′C, α4S6′C, α4L9′C, α4 V13′C, or α4L16′C with wildtype β2. (A) Current trace from an oocyte expressing α4 V13′Cβ2. Two pulses of ACh (100 μM) were applied before and after a 5 min incubation with 2.5 mM MTSEA + and 1 mM ACh. (B) Trace from an oocyte expressing the α4 V13′Cβ2 mutant. Two pulses of ACh (100 μM) were applied before and after a 3 min preincubation with bicyclic alcohol 5 (1 mM), followed by a 5 min incubation of 2.5 mM MTSEA + 1 mM ACh in the presence of bicyclic alcohol 5. (C) Percentage irreversible change in ACh (1 mM) induced currents in three different conformational states of the channel. Open Channel: 2.5 mM MTSEA + 1 mM ACh incubated for 5 min. Closed Channel: 2.5 mM MTSEA incubated in the absence of ACh for 5 min. Desensitized Channel: 1 mM ACh incubated for 1 min, then 1 mM ACh + 2.5 mM MTSEA incubated for 5 min. All mutants were accessible to sulfyhydryl modification in the open and desensitized channel states. Only α4S6′C was not accessible to MTSEA in the closed channel state and possibly the desensitized state. Data are mean ± SEM (n = 3 − 8 oocytes from at least two batches). Statistical significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001 and compares the effects of MTSEA on the cysteine mutant compared to wildtype. (D) Effects of competing bicyclic alcohol 5 before and after MTSEA on the same mutant. Open Channel: 1 mM bicyclic alcohol 5 was preincubated for 3 min and then treated with 1 mM ACh + 2.5 mM MTSEA for 5 min. Closed Channel: 1 mM bicyclic alcohol 5 incubated for 3 min then competed with 2.5 mM MTSEA in the absence of ACh. Desensitized Channel: 1 mM ACh + 1 mM bicyclic alcohol 5 preincubated for 3 min and then treated with 2.5 mM MTSEA. Data are mean ± SEM of N > 3 oocytes from at least two different batches. Statistical significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001 and compares differences between the effects of MTSEA in the presence and absence of bicyclic alcohol 5 on cysteine mutants.