Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity

Abstract

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel plays a central role in protection of cardiac and neuronal cells against ischemia and apoptosis, but its molecular structure is unknown. Succinate dehydrogenase (SDH) is inhibited by mitoK(ATP) activators, fueling the contrary view that SDH, rather than mitoK(ATP), is the target of cardioprotective drugs. Here, we report that SDH forms part of mitoK(ATP) functionally and structurally. Four mitochondrial proteins [mitochondrial ATP-binding cassette protein 1 (mABC1), phosphate carrier, adenine nucleotide translocator, and ATP synthase] associate with SDH. A purified IM fraction containing these proteins was reconstituted into proteoliposomes and lipid bilayers and shown to confer mitoK(ATP) channel activity. This channel activity is sensitive not only to mitoK(ATP) activators and blockers but also to SDH inhibitors. These results reconcile the controversy over the basis of ischemic preconditioning by demonstrating that SDH is a component of mitoK(ATP) as part of a macromolecular supercomplex. The findings also provide a tangible clue as to the structural basis of mitoK(ATP) channels.

Fig. 1.

SDH, mABC1, PIC, ANT, and ATPase interact with each other in co-IP studies on rat liver mitochondrial IM. IM of mitochondria was used as a control. (A) Co-IP of the 30-kDa component of SDH with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. (B) Co-IP of the 70-kDa components of SDH with Abs against 30- and 70-kDa of SDH, mABC1, ANT, ATPase, and PIC. (C) Co-IP of the α-subunit of ATPase with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. The α-subunit of ATPase has a molecular mass of ≈60 kDa. An Ab against the β-subunit of ATPase coimmunoprecipitated the same proteins. (D) Co-IP of PIC with Abs against 30- and 70-kDa component of SDH, ATPase, PIC, and mABC1. PIC has a molecular mass of ≈30 kDa. (E) Co-IP of mABC1 with Abs against 70-kDa components of SDH, ATPase, mABC1, and PIC. mABC1 has a molecular mass of ≈55 kDa. Negative controls included Abs against complex I and IV of the respiratory pathway, Kir6.1, and cyclophilin D. The IM of mitochondria was prepared in 1.2% digitonin and 6% lubrol WX and was dissolved in 300 mM KPi/10% ethylene glycol/5 mM EDTA/4 mM ATP/0.5 mM DTT, pH 7.9. The columns were also washed with 2–3% (octylphenoxy)polyethoxyethanol to increase the specificity of the interactions between proteins.

Fig. 2.

Silver staining and Western blot analysis of the purified M-fraction. (A) Silver-stained SDS/PAGE gel of the mitochondrial IM extract and the purified M-fraction. We loaded ≈150 and 40 μg of protein in the IM and M-fraction lanes, respectively. (B) Western blots of the M-fraction with Abs against the 30- and 70-kDa components of SDH, PIC, α-subunit of ATPase, ANT, and mABC1. We loaded 40 μg of the M-fraction in each lane. The Ab against mABC1 also recognizes a smaller ≈30-kDa band.

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.

Fig. 4.

An inhibitor of SDH (3-NPA) activates the channel, whereas 5-HD reverses this effect. (A) Effects of 3-NPA on K⁺ transport in proteoliposomes. The K⁺ transport was significantly activated by addition of 1 mM 3-NPA, whereas 5-HD reversed this effect. (B) A similar response to 3-NPA was noted in lipid bilayer. Microsomes from the M-fraction were incorporated into lipid bilayer, and single K⁺ channel activity was recorded in the absence of any modulator. After addition of 1 mM of 3-NPA, the P_o increased ≈4-fold. Addition of 5-HD reversed this activation significantly. Closed and open current levels for all recordings are indicated by solid and dashed horizontal lines, respectively. (C) Summary of studies on 3-NPA in lipid bilayer.