Single amino acid mutations in transmembrane domain 5 confer to the plasma membrane Ca2+ pump properties typical of the Ca2+ pump of endo(sarco)plasmic reticulum.

Conserved residues in some of the transmembrane domains are proposed to mediate ion translocation by P-type pumps. The plasma membrane Ca(2+) pump (PMCA) lacks 2 of these residues in transmembrane domains (TM) 5 and 8. In particular, a glutamic acid (Glu-771) residue in TM5, which is proposed to be involved in the binding and transport of Ca(2+) by the sarcoplasmic reticulum Ca(2+) pump (SERCA), is replaced by an alanine (Ala-854) in the PMCA pump. Ala-854 has been mutated to Glu, Asp, or Gln; Glu-975 in TM8, which is an Ala in the SERCA pump, has been mutated to Gln, Asp, or Ala. The mutants have been expressed in three cell systems, with or without the help of viruses. When expressed in large amounts in Sf9 cells, the mutated pumps were isolated and analyzed in the purified state. Two of the three TM8 mutants were correctly delivered to the plasma membrane and were active. All the TM5 mutants were retained in the endoplasmic reticulum; two of them (A854Q and A854E) retained activity. Their properties (La(3+) sensitivity and decay of the phosphorylated intermediate, higher cooperativity of Ca(2+) binding with a Hill's coefficient approaching 2) differed from those of the expressed wild type PMCA pump, and resembled those of the SERCA pump.

Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit.

The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.

Electrophysiological characterization of ionic transport by the retinal exchanger expressed in human embryonic kidney cells.

The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity.

The organization of the human gene NCX1 encoding the sodium-calcium exchanger.

The intron-exon organization of the human Na/Ca-exchanger gene NCX1 and of the N-terminal half of the related gene NCX2 has been determined. The NCX1 gene consists of 12 exons spread over 200 kb on chromosome 2 close to STS D2S2328 and encodes a 6.2-kb transcript. Both NCX1 and NCX2 feature an unusual 1.8-kb exon, containing two-thirds of the protein coding sequence and a similar area of the coding sequences split into several small exons, displaying tissue-specific alternative splicing. The similar intron positions in the "cardiac" (NCX1) and "brain" (NCX2) mammalian exchanger genes suggest their origin from the recent duplication and translocation of a common ancestral gene, a putative precursor of which has been identified in the nematode Caenorhabditis elegans.

Expression of Na(+)-Ca2+ exchanger with modified C-terminal hydrophobic domains and enhanced activity.

A 6-Kb canine cDNA fragment complementary to the 5' region of the 7-Kb mRNA encoding the cardiac Na+-Ca2+ exchanger was expressed in human kidney 293 cells. The mRNA products were reverse transcribed and amplified by PCR. The determined DNA sequence of the amplified DNA fragments revealed the presence of an intron that was alternatively spliced. The partial exon sequence, located at the 3' end of the 6-Kb cDNA, was alternatively connected to bases 3198, 2821, 2620 and 1844 in four types of splicing products identified. In the largest product the adjoining exon was located after the putative stop codon of the regular sequence. In a second and third type of shortened transcripts, a hydrophobic sequence encoded by the spliced-in exon was linked with the 4th or the 5th extracellular loops, and could possibly replace transmembrane segments 9 or 11. In the fourth type of spliced transcript the in-frame exon sequence introduced one Leu followed by a stop codon in the large hydrophilic loop. Measurements of Ca2+ uptake in 293 cells expressing the modified exchanger indicated a higher activity in comparison with 293 cells expressing the 3.7-Kb cDNA, in which this alternative splicing does not occur. Deletion mutagenesis of the C-terminal region encoded by the spliced-in exon was performed to investigate its role in the enhancement of the transport activity.

Tissue distribution of the four gene products of the plasma membrane Ca2+ pump. A study using specific antibodies.

Antibodies against the four isoforms of the human plasma membrane Ca(2+)-ATPase (PMCA) were raised using an N-terminal sequence of the pump as epitope. The antibodies against PMCA isoforms 1, 2, and 3 were not species-specific, e.g. they also recognized the corresponding proteins in rat, whereas that against the human PMCA isoform 4 failed to do so. The tissue distribution of the four isoforms was estimated by Western blot analysis. Two, PMCA1 and PMCA4, were expressed in all tissues tested (with the exception of the choroid plexus, where the former was not detected). In most tissues the signal from the PMCA1 protein exceeded that of PMCA4, the exception being the erythrocyte. The PMCA2 and PMCA3 proteins were only found in neuronal tissues; the PMCA2 protein was present in high concentrations in the cerebellum and in the cerebral cortex. At variance with previous results on mRNA (e.g. the kidney) no other tissues contained the PMCA2 protein. PMCA3 was the other tissue-specific isoform; in agreement with results in the rat, the protein was found in human neuronal tissues, particularly in the choroid plexus, but was practically absent in all other tissues tested.

An alternative splicing site modifies the carboxyl-terminal trans-membrane domains of the Na+/Ca2+ exchanger.

The 6-kilobase (kb) cDNA of pTB11 clone and its 5' fragment of 3.7 kb encoding the canine heart Na+/Ca2+ exchanger (Nicoll, D.A., Longoni, S., and Philipson, K.D. (1990) Science 250, 562-565) were transiently expressed in 293 cells to investigate the role of the 3'-"untranslated" region. Both fragments yielded high levels of expressed protein that were well incorporated in the membranes. Cells expressing the 6-kb cDNA produced rearranged transcripts of smaller than expected size. A 120-kDa polypeptide was produced in cells expressing the modified exchanger, and Ca2+ uptake was higher in this type of transfected cells. A constant stretch of nucleotides located at the 3' end of the 6 kb cDNA was found to be connected, by alternative RNA splicing, to four different upstream sequence positions. The deduced hydrophobic sequence of the spliced-in exon could replace the IX or the XI trans-membrane domain of the exchanger protein in two spliced isoforms. The new exon sequence was not completely included in the pTB11 insert, i.e. these two products were artificially truncated. The RNA processing of these two alternative 5'-splicing sites also occurred in tissues, as shown by RNase protection analysis. In a third type of isoform the splicing took place downstream of the originally proposed stop codon, whereas in a fourth type a stop codon was introduced after the V hydrophobic segment in the large intracellular loop.

Multiple divergent mRNAs code for a single human calmodulin.

The isolation of a novel complementary DNA (cDNA) clone coding for human calmodulin (CaM) is reported. Although it encodes a protein indistinguishable from the only known higher vertebrate calmodulin, its nucleotide sequence varies extensively from that of two previously reported human CaM cDNAs (Wawrzynczak and Perham, 1984; SenGupta et al., 1987). Only 82 and 81% identity, respectively, is found between the newly isolated and the two known human mRNAs in their coding regions. No striking homology is present in their noncoding regions. Codon usage in the three CaM mRNAs is also surprisingly divergent. A 2.3-kilobase mRNA corresponding to the newly isolated clone is expressed to varying extents in several human tissues, together with an approximately 0.8-kilobase mRNA species presumably arising from alternative polyadenylation of the same primary transcript. The results indicate that the human genome contains at least three divergent CaM genes that are under selective pressure to encode an identical protein while maintaining maximally divergent nucleotide sequences. Partial characterization of a genomic clone specifying the 3' portion of the newly identified CaM mRNA shows that this gene contains introns at identical positions as the previously characterized bona fide vertebrate CaM genes. Evolutionary implications of the presence of a CaM multigene family are discussed.

The fatty acid composition of subcellular membranes of rat liver, heart, and brain: diet-induced modifications.

The influence of diets having different fatty acids composition on the fatty acid content of (the phospholipids) of rat liver mitochondria and microsomes, heart mitochondria, brain mitochondria and microsomes has been analyzed. It has been found that each organelle has its own peculiar composition in fatty acids. This composition may be profoundly influenced by the diet, but to different degrees in different organelles. Those of brain are most resistant. The changes observed are rather rapid, being generally already maximal after three weeks of treatment. The parallel between fatty acid composition of diets, and the changes observed in the organelles, is not strict, and is probably influenced by the metabolic competition among oleic acid, linoleic acid, linolenic acid. Unusual fatty acids like erucic acid, trans-oleic acid, and trans-linoleic acid can also become incorporated into the membranes of cell organelles.

Hydroperoxides can modulate the redox state of pyridine nucleotides and the calcium balance in rat liver mitochondria.

When rats are fed a selenium-deficient diet, the glutathione peroxidase activity in liver mitochondria decreases within 5 weeks to 0-6% of that of control animals fed on a diet supplemented with 0.5 ppm of selenium as sodium selenite. Analysis of the temperature dependence of energy-linked Ca(2+) uptake by means of Arrhenius plots reveals two breaks (at around 11 degrees C and 24 degrees C) in mitochondria isolated from selenium-supplemented animals, whereas in selenium-deficient rats the break at 11 degrees C is absent. Ca(2+)-loaded mitochondria of selenium-supplemented rats-i.e., with active glutathione peroxidase in the matrix-lose Ca(2+) rapidly, with a concomitant oxidation of endogenous NAD(P)H, when exposed to t-butyl hydroperoxide or H(2)O(2). In contrast, in selenium deficiency, t-butyl hydroperoxide and H(2)O(2) induce neither a release of Ca(2+) nor an oxidation of NAD(P)H. The peroxide-induced oxidation of NAD(P)H is reversible in the presence of succinate when no Ca(2+) has been taken up. When Ca(2+) has previously been accumulated, however, the oxidation of NAD(P)H is irreversible. Enzymatic analysis of mitochondrial pyridine nucleotides reveals that the peroxide-induced oxidation of NAD(P)H in Ca(2+)-loaded mitochondria leads to a loss of NAD(+) and NADP(+). It is proposed that the redox state of mitochondrial pyridine nucleotides can be or is in part controlled by glutathione peroxidase and glutathione reductase and is a factor in the balance of Ca(2+) between mitochondria and medium.