Figure 6. Single channel assay of mSlo (left traces) and mSlo-GFP (right traces) with Barium and charybdotoxin (CTX).

Channels were expressed in COS-7 cell membranes and incorporated into lipid bilayers. Channel openings are upward, and the small arrow to the left of each trace denotes the closed state. All traces represent single channel currents in symmetrical 150 mM solutions at +30 mV. Top traces represent control experiments with no CTX. An expanded time scale is shown in the Traces second from the top represent a portion of the trace above with a 100 fold expanded time scale. From 23 minutes of recorded data for CTX block of mSlo-GFP, the burst time constant was 3.12 sec and the blocked time constant was 13.1 sec, which corresponds to an on rate of 12.8 x 106 s-1 M-1 and an off rate of 76.3 x 10-3 s-1, respectfully. These data give a CTX equilibrium constant KD of 6.0 nM for this channel. These data closely match the single channel diplayed for mSlo, which has a CTX equilibrium constant KD = 6.7 nM. For overall kinetic parameters from a total of 12 experiments, see table 1. The Barium block of mSlo-GFP shows a blocked time constant of 4.56 sec, almost exactly equal to mSlo (4.64 sec).

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