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To provide experimental evidence for the topology of the Na+-Ca2+ exchanger protein NCX1 in the membrane, indirect immunofluorescence studies using site specific anti-peptide antibodies and Flag-epitope insertion into chosen locations of the protein were carried out. Anti-peptide antibodies AbO-6 and AbO-8 were raised against peptide segments present in a large hydrophilic loop of about 500 amino acids, which separates the hydrophobic amino terminal part of the protein from the hydrophobic carboxy terminal. AbO-10 was raised against the C-terminal tail of the protein. All three antibodies bound to the exchanger protein expressed in transfected cells, in rat brain synaptic plasma membrane and in dog sarcolemmal preparations. The antibodies bound only to those NCX1 isoforms that contained the epitope against which they were raised. Detection of the exchanger protein in transfected cells in situ required the addition of permeabilizing agents suggesting an intracellular location of the epitopes to which AbO-6, AbO-8 and AbO-10 bind. The Flag epitope was inserted into ten putative extramembraneous segments along the exchanger protein. For topology studies, only the Flag-mutants that retained Na+-Ca2+ exchange activity in whole HeLa cells, were used. Immunofluorescence studies indicated, that the N-terminal of the protein is extracellular, the first hydrophilic loop separating transmembrane helices 1 and 2 as well as the C-terminal, are intracellular.

A ft~n~tjon~l rat heart Na"-Ca" exchanger gene has been obtained by splicing and ligating two partially overlapping clones isolated from a rat heart /ZZAP cDNA library. The deduced primary structure of the protein encoded by the open reading frame corresponds to 971 amino acids, that can be organized into 12 transmembrane helices. The cloned gene was functionally expressed in HeLa cells. Maximal expression was detected 18 h after transfection, after which transport activity rapidly declined. The electrogenic properties of the cloned transporter were demonstrated following reconstitution of the expressed exchanger protein into a tightly sealed phospholipid membrane.

The rat brain Na(+)-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al. (14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide (N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site (N26), and the entire putative signal peptide and putative signal peptidase cleavage site (N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108% (N21), 37.2% (N26), and 60.06% (N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 --> Asp, resulted in a decrease in Na(+)-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1% (N21), 26.75% (N26), 85.36% (N31), and 31% (Ala-32 --> Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 --> Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na(+)-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 -->Asp. The amino-terminal truncated mutants of the cloned Na(+)-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na(+)-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.

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Mudskippers build burrows on the mudflat in mangrove swamps ofestuaries. Water within these burrows is usually low in oxygen content (Gordon et a/., 1978). Working with the local mudskipper, Periophthalmus chrysospilos (Bleeker, I853), Chew et al. (1990) observed that its oxygen consumption rate changed as a function of dissolved oxygen content of the ambient sea water. At 1~1 ml-l of dissolved oxygen, the respiratory rate was approximately one quarter that of the normoxic control fish. In addition, it could survive for at least 6 h in water containing 0.8 pl ml-' of dissolved oxygen. Its respiratory rate decreased sharply within the first 5 min of exposure and remained low throughout the 6 h experimental period. Oxygen consumption of these hypoxic fish increased only slightly upon recovery in normoxic sea water. Hence, Chew el al. (1990) suggested that P. chrysospilos might be able to tolerate environmental hypoxia by reducing its metabolic rate. In order to confirm this interesting finding, the present studies were undertaken to examine the effects of environmental hypoxia on the energy charge and the contents of glycogen and various metabolites in the muscle of this fish. Experiments were also performed with fish exposed to functional hypoxia for comparison. P. chrysospi/os (5-14 g body weight) were collected along the shore near the estuarine canal at Pasir Ris, Singapore and maintained in the laboratory at 25" C in 50% sea water. They were exposed to 50% sea water containing 0.8 pl ml-l of dissolved oxygen at 25" C for 6 h as described by Chew et a / . (1990). Ten minutes before the end of the exposure period, the fish were anaesthetized by introducing 0.004% (w/v) MS 222 (Sigma, U.S.A.) into the container. They were then killed quickly by a blow on the head. Fish were exposed to functional hypoxia by locomotory exertion to exhaustion. They were ' chased ' individually in small aquaria (27 x 17 x 19 cm) for 5-10 min until they became inert to further mechanical stimulation. They were then caught and killed immediately. Control fish were anaesthetized in MS 222 for 10 min before being killed. The lateral muscles of the dead fish were excised as quickly as possible. No attempt was made to separate the red and white muscles. The samples were immediately freezeclamped in liquid nitrogen with pre-cooled aluminium tongs (Faupel et al., 1972). The whole procedure took less than 25 s. The frozen sample was homogenized and centrifuged according to the method of Chew et al. (1990). An aliquot of the supernatant fluid was diluted appropriately for the quantitative determination of lactate, malate, citrate and pyruvate by an Eyela Carboxylic Acid Analyzer S-14 (Tokyo Rikakika Co., Japan). The remaining of the supernate was titrated with K,CO, ( 0 . 3~) to pH 6.5-7.0 for further creatine phosphate (CrP, Lamprecht et al., 1974), adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP, Scheibel et al., 1968) analyses. Spectrophotometric analyses were performed with a Shimadzu UV-260 62...

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