Figure 6.

Barium slows MTSEA modification of A384C in the open but not closed state. (A) Ba²⁺ protection of open state modification. Top traces from control patch in asymmetrical K⁺ at 0 mV successively exposed to MTSEA for 1 s followed by washout in 2 μM Ca²⁺. Bottom traces from an experimental patch in which the channel was blocked with 100 μM Ba²⁺ for 1 s, and subsequently exposed to MTSEA with Ba²⁺. After MTSEA washout for 1 s Ba²⁺ was washed out to assess current amplitude. (B) Time course of MTSEA modification in control (closed symbol) and with Ba²⁺ block (open symbol). Lines represent a single exponential fit with time constant of 3.6 and 6.7 s for control and Ba²⁺ block modification time course, respectively. (C) Closed state MTSEA modification. Top traces from a control patch in which channels were closed for 2 s, MTSEA applied in the closed state for 5 s, and the channels washed for 2 s in “0” Ca²⁺ solution before opening to monitor the fraction of current modified. This procedure was repeated until modification was complete. Bottom traces from an experimental patch measuring closed state MTSEA modification with Ba²⁺ trapped in the pore. Open channels were first blocked with 100 μM barium, closed in the presence of barium, Ba²⁺ washed out, then 200 μM MTSEA applied for 1 s. MTSEA was washed out in the closed state for 2 s before reopening the channels in 2 μM Ca²⁺. This procedure was repeated until modification was complete. For clarity the figure shows only the first application, then every fifth application. (D) Time course of MTSEA modification of A384C in the absence (open circles) or presence (closed circles) of Ba²⁺. Lines represent a single exponential fit with time constant of 13.8 and 16.2 s to control and Ba²⁺ block modification time course, respectively. (E) Bar graph of mean MTSEA modification rate ± SEM for open and closed state in the absence and presence of Ba²⁺ block. Asterisk indicates P < 0.01. Concentration of MTSEA in all experiments was 200 μM.