0 1990 by The Amencan Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 18, Issue of June 25, pp. 105!22-10526, 1990 Printed in U.S. A. Reconstitution of the Beef Heart and Rat Liver Mitochondrial K+/H+ (Na+/H+) Antiporter QUANTITATION OF K’ TRANSPORT WITH THE NOVEL FLUORESCENT PROBE, PBFI* (Received for publication, September 15, 1989) Petr JeiekS, Fakhri Mahdi, and Keith D. GarlidO From the Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699 New indicators for fluorescent measurement of Na+ and K+ ions should prove particularly useful for studies of reconstituted carriers of these ions. We show that PBFI, a K’-specific probe, provides a convenient and sensitive assay for the study of K’ uptake mediated by the reconstituted mitochondrial K’/H+ (Na+/H+) anti- porter. Fluorescent measurements have enabled us for the first time to establish reconstitution of the K’/H’ (Na’/H’) antiporter from beef heart as well as from rat liver mitochondria. This technique has also enabled us to establish that dicyclohexylcarbodiimide is capable of complete inhibition of K’/H’ antiport in the recon- stituted system, in accord with findings in intact mi- tochondria. PBFI fluorescence, which measures net K+ uptake, was essential for this corroboration, since di- cyclohexylcarbodiimide is not capable of complete in- hibition of 42K+/K’ or 86Rb’/Rb’ exchange, presumably because it acts selectively on proton transport within the carrier. Mitochondrial volume homeostasis is maintained by the regulated functioning of a nonselective K’/H’ (Na+/H+) an- tiporter which transports all alkali cations (l-4). This carrier is inhibited reversibly by quinine and other amphiphilic amines (l-5) and irreversibly by DCCD’ (3,4). Reconstitution of the non-selective K’/H’ (Na’/H+) antiporter from rat liver mitochondria was recently established using 86Rb+ uptake assays (6). This assay has several disadvantages, the most important of which is that it cannot distinguish between Rb+/ Rb’ and Rb’/H’ translocation modes. Thus, Rb’/Rb’ ex- change is only partially inhibited by DCCD, an effect that has been demonstrated in both intact mitochondria and in proteoliposomes (6, 7). On the other hand, net cation trans- port by the antiporter (cation/proton exchange) is completely inhibited by DCCD, an effect that has only been demonstrated in intact mitochondria (3, 4). For this reason, it has not yet been established whether DCCD is capable of inhibiting net * This research was supported in part by Grants GM 31086, HL 43814, and HL 36573 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “oduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. t Visiting Fellow. Permanent address: Institute of Physiology, Czechoslovak Academy of Sciences, Videhski 1083, 14220 Prague, Czechoslovakia. § To whom correspondence and reprint requests should be ad- dressed. ’ The abbreviations used are: DCCD, N,N’-dicyclohexylcarbodi- imide; SMPs, submitochondrial particles; TEA, tetraethylammonium cation; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CCCP, carhonyl cyanide m-chlorophenylhydrazone. cation transport via the reconstituted antiporter. We have now explored the use of a novel potassium flu- orescent indicator, PBFI. This technique has allowed us to obtain evidence for reconstitutive activity of the K+/H+ (Na+/ H+) antiporter from beef heart mitochondria as well as from rat liver mitochondria, using the physiological substrate, K’. We can now demonstrate complete inhibition of K+/H+ an- tiport by DCCD in the reconstituted system, since this tech- nique senses net K’ uptake. Finally, we demonstrate that propranolol and timolol inhibit K+/H+ exchange in the recon- stituted heart preparations with 1,, values similar to those observed in intact mitochondria (5). EXPERIMENTAL PROCEDURES Detergent Solubilization of Membrane Proteins-SMPs from beef heart, prepared according to Ref. 8, and from rat liver, prepared by sonication (9), were isolated by centrifugation. They were resus- pended at 50 mg of protein/ml in 30 mM TEA/SO,, and 1 mM TEA/ EDTA. 10 mM TEA/HEPES. DH 7.2 (Buffer A). and the stock . ., .I suspensions were frozen and stored at -70 “C. Thawed stock was diluted to 9.5 mg/ml with Buffer A containing Triton X-100 (3.3 mg/ ml protein) and beef heart cardiolipin (3 mg/ml protein). The sus- pension was stirred for 20 min at 0 “C. Extracted proteins were then separated from unsolubilized material by centrifugation at 130,000 x g for 35 min. Preparation of Liposomes and Reconstitution of Membrane Ex- tracts-Liposomes were prepared essentially as described by Kakar et al. (6):116 mg of asoleciin (crude soybean phosphatidylcholine tvne IV. Siemal was dried and disDersed in 1.65 ml of 30 mM TEA/ &k, 1 &M’TEk/EDTA, 120 mM -kA/HEPEs, pH 7.4 @Lffer Bj. The fluorescent K’ indicator, PBFI (Molecular Probes, Inc.), was added to a final concentration of 65-75 pM. The lipid suspension was then sonicated for about 3 min in a batch sonicator. 1.5 ml of the sonicated liposome suspension was mixed with 1.5 ml of a crude protein extract or with an identical solution lacking proteins. 200-~1 aliquots of this mixture were placed on the tops of l- ml Bio-Beads SM-2 (Bio-Rad) columns which had been pre-equili- brated with a 1:l mixture of Buffers A and B. The columns were allowed to stand at room temperature for 60 min and then centrifuged at 400 x g using a Sorval GLC-1 tabletop centrifuge. The proteoli- posomes were washed free of external probe by passage through l-ml SeDhadex G-25-300 (Siema) columns that had been me-eauilibrated with a 1~1 mixture of Buffers A and B. The “internal medium” thus contains 30 mM TEA/SO,. 1 mM TEA/EDTA. 65 mM TEA/HEPES. pH 7.4, and 65-75 g’M PBFI. The final proteoliposome suspension usually contained 55 mg of lipid/ml and 630 fig of extracted protein/ ml. Internal volume of proteoliposomes was estimated from the distri- bution of [14C]TEA’ in parallel samples to which [“C]TEABr was added before Bio-Beads treatment. Duplicate 50-&l aliquots were counted prior to Bio-Beads treatment &d after washing out the external radioisotope by Sephadex (see above). The derived internal volume usually amounted to 0.2 &‘mg of lipid in proteoliposomes and 1.1 pl/mg of lipid in liposomes. The amount of protein actually reconstituted into the vesicles was evaluated by the Amido Black method (10). Fluorescence Measurements-Fluorescence of PBFI-loaded proteo- 10522 by guest, on October 16, 2011 www.jbc.org Downloaded from