Fig. 3.

Analysis of the M-fraction for mitoK_ATP channel activity in proteoliposomes and lipid bilayer. (A) Changes in PBFI fluorescence in proteoliposomes containing the M-fraction and nonreconstituted liposomes. Proteins in the M-fraction were reconstituted into liposomes and fluorescence of PBFI marker was measured. There is a higher rate of increase in fluorescence in proteoliposomes compared with nonreconstituted liposomes. The rate of increase in fluorescence is directly proportional to the transport of K⁺ into the proteoliposomes. Cation selectivity was confirmed by using the Na-sensitive 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-tetraammonium salt (SBFI) and replacing K with Na ions (data not shown). (B) The K⁺ transport into the proteoliposomes was significantly activated by 100 μM diazoxide. mitoK_ATP inhibitors, 5-HD (500 μM), glybenclamide (10 μM), and ATP (2 mM), all inhibited diazoxide activated K⁺ transport activity. Lower concentrations of ATP (i.e., 200 μM) also resulted in a decrease in K⁺ transport activity. (C) The functional properties of the M-fraction were also studied in lipid bilayers. Unitary K⁺ currents recorded in lipid planar bilayers after fusion of native microsomes from the M-fraction. Single K⁺ channel activity was recorded during the application of a continuous voltage ramp from -30 to +30 mV during 5 s. Channel openings are shown as inward deflections and represent K⁺ movement. Nonspecific leak current was subtracted, and the unitary conductance value was determined as the slope of a linear fit to the open level. (D) K⁺ channel activity was recorded before and after addition of 100 μM diazoxide. (E) To study the effects of mitoK_ATP inhibitors, 100 μM diazoxide was added to activate the channel, followed by the addition of 500 μM 5-HD, 10 μM glybenclamide, or 2 mM ATP. These reagents all resulted in a significant inhibition of the diazoxide-activated channel activity. Total-amplitude histograms constructed from 3 min of continuous recording in each condition are also shown. Multipeak Gaussian curves were fitted to the histograms. Mean amplitudes were defined from the difference between peaks. P_o values were determined from the open/total area ratio. Top represents a representative experiment in the presence of diazoxide. (F) Summary of the lipid bilayer studies. Each experiment was performed at least three times. ΔF, Change in fluorescence; Gly, glybenclamide; Atr, atractyloside.