Figure 2.
Figure 2. G45C and V38C residues are modified by MTSEA–biotin-X. Segments of patch clamp records of G45C and V38C after the addition of MTSEA–biotin-X to the bath solution at a final concentration of 1 or 0.5 mM. Arrows at the bottom of each panel mark step changes in conductance that are consistent with the reaction of a cysteine residue with MTSEA–biotin-X. Changes in current levels were determined with the all-point histograms shown on the right side of each panel. (A) Inside-out recording of two G45C channels. Nine conductance changes are evident, consistent with modification of 9 of 12 available G45C residues. The first modification event occurred 20 s after the bath application of 1 mM MTSEA–biotin-X. (B) Outside-out recording of a single G45C channel illustrating five conductance changes, consistent with modification of five of six available cysteine residues. The first modification event occurred 6 s after the bath application of 1 mM MTSEA–biotin-X. (C) Inside-out recording of a single V38C channel illustrating four conductance changes consistent with modification of four of six available subunits. The first modification event occurred 18 s after the bath application of 1 mM MTSEA–biotin-X. (D) Outside-out recording of a single V38C channel illustrating six conductance changes. It is not clear if the final conductance change, marked by an asterisk, resulted from a reaction, gating event, or channel loss; thus, at least five of six cysteine residues are modified. The first modification event occurred 64 s after the bath application of 0.5 mM MTSEA–biotin-X. The longer time to the first modification event correlates with the lower concentration of MTSEA–biotin-X used in this record (0.5 mM vs. 1.0 mM) than was used in the previous records. (D1) This is an enlargement of the boxed region of D. The event marked by the single asterisk is a Vj-gating transition that occurred after modification of two cysteine residues. The event marked by two asterisks is a loop-gating transition that occurred after three cysteine subunits were modified by MTSEA–biotin-X. Note that the loop- or slow-gating event involves a series of small amplitude transitions giving the appearance of a complex gating event with a measurable time constant, whereas the Vj-gating event occurs as a single fast step between the open state and a subconductance state. Zero current level indicated by the dashed line is the leak-subtracted current.

G45C and V38C residues are modified by MTSEA–biotin-X. Segments of patch clamp records of G45C and V38C after the addition of MTSEA–biotin-X to the bath solution at a final concentration of 1 or 0.5 mM. Arrows at the bottom of each panel mark step changes in conductance that are consistent with the reaction of a cysteine residue with MTSEA–biotin-X. Changes in current levels were determined with the all-point histograms shown on the right side of each panel. (A) Inside-out recording of two G45C channels. Nine conductance changes are evident, consistent with modification of 9 of 12 available G45C residues. The first modification event occurred 20 s after the bath application of 1 mM MTSEA–biotin-X. (B) Outside-out recording of a single G45C channel illustrating five conductance changes, consistent with modification of five of six available cysteine residues. The first modification event occurred 6 s after the bath application of 1 mM MTSEA–biotin-X. (C) Inside-out recording of a single V38C channel illustrating four conductance changes consistent with modification of four of six available subunits. The first modification event occurred 18 s after the bath application of 1 mM MTSEA–biotin-X. (D) Outside-out recording of a single V38C channel illustrating six conductance changes. It is not clear if the final conductance change, marked by an asterisk, resulted from a reaction, gating event, or channel loss; thus, at least five of six cysteine residues are modified. The first modification event occurred 64 s after the bath application of 0.5 mM MTSEA–biotin-X. The longer time to the first modification event correlates with the lower concentration of MTSEA–biotin-X used in this record (0.5 mM vs. 1.0 mM) than was used in the previous records. (D1) This is an enlargement of the boxed region of D. The event marked by the single asterisk is a Vj-gating transition that occurred after modification of two cysteine residues. The event marked by two asterisks is a loop-gating transition that occurred after three cysteine subunits were modified by MTSEA–biotin-X. Note that the loop- or slow-gating event involves a series of small amplitude transitions giving the appearance of a complex gating event with a measurable time constant, whereas the Vj-gating event occurs as a single fast step between the open state and a subconductance state. Zero current level indicated by the dashed line is the leak-subtracted current.

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