Figure 6. Inhibition of L70A/N8 homo-... | Rockefeller University Press

Figure 6.

$Figure 6. Inhibition of L70A/N8 homo- and heterochannels by external TEA. (A) Electrophoretical separation of five L70A/N8 tetramers. The coexpressing S35-labeled proteins were run on a 12.5% SDS–polyacrylamide gel for 16 h. Lanes A through E were tetramers formed at L70A/N8 plasmid ratios of 4:0, 3:1, 2:2, 1:3, and 0:4. The identified five bands corresponded to all subunit combinations, L70A4-nN8n, where n is the number of N8 subunits in the tetramer, n = 0, 1, 2, 3, and 4. Although there is some overlap in the bands, the lack of any overlap in the data collected using excised tetramers indicates that the correct single tetramers were used for the experiments in B and C, and each type of tetramer has specific conductance, as reported previously (Tan et al., 2010). (B) Current traces for L70A/N8 co-tetramers with five-subunit combinations purified from the gel in A (−60 mV). For each tetramer, the top trace was recorded without TEA and the bottom trace with external TEA at a concentration around IC50. (C) TEA concentration–dependent inhibition fraction for the five L70A/N8 co-tetramers. Data points are presented as mean ± SD. (D) TEA binding energy ΔG for each type of L70A/N8 co-tetramer, as calculated from IC50.$

Inhibition of L70A/N8 homo- and heterochannels by external TEA. (A) Electrophoretical separation of five L70A/N8 tetramers. The coexpressing S³⁵-labeled proteins were run on a 12.5% SDS–polyacrylamide gel for 16 h. Lanes A through E were tetramers formed at L70A/N8 plasmid ratios of 4:0, 3:1, 2:2, 1:3, and 0:4. The identified five bands corresponded to all subunit combinations, L70A_4-nN8_n, where n is the number of N8 subunits in the tetramer, n = 0, 1, 2, 3, and 4. Although there is some overlap in the bands, the lack of any overlap in the data collected using excised tetramers indicates that the correct single tetramers were used for the experiments in B and C, and each type of tetramer has specific conductance, as reported previously (Tan et al., 2010). (B) Current traces for L70A/N8 co-tetramers with five-subunit combinations purified from the gel in A (−60 mV). For each tetramer, the top trace was recorded without TEA and the bottom trace with external TEA at a concentration around IC₅₀. (C) TEA concentration–dependent inhibition fraction for the five L70A/N8 co-tetramers. Data points are presented as mean ± SD. (D) TEA binding energy ΔG for each type of L70A/N8 co-tetramer, as calculated from IC₅₀.