Mitochondria were isolated as described and suspended at a concentration of 1 mg protein/ml in respiration buffer containing 2.5 mg/ml of BSA. O₂ consumption was measured using a Clark-type O₂ electrode in a thermostatic chamber at 25°C. (A) Representative respiration traces: Following the addition of mitochondria, Left trace: 10 mM Pyruvate plus 5 mM Malate (Pyr+Mal) were added to induce state 2 respiration, 200 µM ADP to induce state 3, and 1 µg/ml oligomycin (Oligo) to induce state 4. Right trace: 10 mM Succinate (Succ) was added followed by 200 µM ADP. (B) Quantitation of the respiratory control ratio (state 3 divided by state 4) from several experiments as shown in A. Data are means ± SEM, N>8.

(A): Osmotic swelling assay for determination of the mK_ATP channel activity. Representative swelling trace of mitochondria (~0.25 mg/ml) in K⁺ media. Where indicated, 1 mM ATP, 10 µM diazoxide (DZX, a K_ATP opener), or 300 µM 5-hydroxydecanoate (5-HD, a K_ATP antagonist) were present in the media. (B): Magnitude of swelling, relative to the control, as determined by a decrease in A₅₂₀ after 0.2 min. incubation in K⁺ media (filled bars) or in Na⁺ media (open bars). (C): The pharmacological profile of the C. elegans mK_ATP channel demonstrated in response to activators and inhibitors in K⁺ media. Chromakalim (Crmk), pinacidil (Pncd) and malonate (Malo) are all K_ATP openers, while 5-HD and glyburide (Gly) are K_ATP antagonists. The 3 conditions for each opener (i.e. opener alone and with 2 antagonists) are shaded the same. Control swelling after 0.2 min resulted in a decrease in A₅₂₀ of 0.0233 ± 0.0013 OD units in K⁺ media and 0.0167 ± 0.004 OD units in Na⁺ media. Experimental conditions are listed below the x-axis. Data are means ± SEM *p<0.05 vs. control, **p<0.05 vs. ATP, ***p<0.05 vs. ATP+channel activator; N≥4.