Coexpression of mutant channels forms new conductance levels.

Plasmids for E2117K and E2117D were co-transfected and each contained an IE element that produced a different fluorescent marker to ensure that the cells under study expressed both proteins. We used mCherry to tag the expression of E2117D and GFP for E2117K. (A) (Left) is an example of single channel currents from a cell-attached patch with discrete conductance levels as indicated. The stimulus was 4 seconds at -10 mmHg. A potassium bath set the membrane potential near 0 mV and the applied membrane voltage was -50 mV. (A) (Right) are possible combinations of heteromers from subunits. Blue refers to E2117D and orange is E2117K. (B) is the amplitude histogram with three different current levels indicated. The highest level is monomeric E2117D (blue line). Only two new levels appeared (red and green lines). (C) is a bar graph of the three conductance levels (green, red, and blue). These data are the mean responses from 5 different patches with different ratios of transfected DNA. Each patch was stimulated with suction pulses ranging from -10 to -30 mmHg for 2 to 10 seconds at potentials from -50 to -100mV.