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The Na/Ca exchanger has been examined with respect to its molecular biology, its cellular function, and its role in excitation-contraction coupling. The Na/Ca exchanger plays a central part in excitation-contraction coupling, setting the level of sarcoplasmic reticular calcium and contributing to the triggering of sarcoplasmic reticular calcium release. Functional biophysical studies with isolated single cells and caged calcium provide evidence that the Na/Ca exchanger works as a two step sequential transporter. In the heart there are about 250 exchangers.mu-2, operating at a turnover rate of up to about 2500.s-1, with the exchanger carrying -2.56 charges under normal conditions. The Na/Ca exchanger has been recently cloned from diverse mammalian species and several tissues and is largely conserved. It is clear, however, that the function of the Na/Ca exchanger is different in the different tissues. Thus work is in progress in several laboratories, including ours, to determine how the Na/Ca exchanger achieves its tissue specific function. Several modulatory motifs have been seen in studies of the exchanger that may explain some of the tissue specific differences. Interestingly the modulation of the Na/Ca exchanger (for example, by protons, sodium, calcium, ATP, calmodulin) seems to arise from interactions with the intracellular loop.

Antibody based manipulation of the CD137 (4-1BB) co-signaling pathway is an attractive option for the treatment of cancer and autoimmune disease. We developed a chimeric anti-human CD137 monoclonal antibody (GG) and characterized its function. As a component of planned preclinical studies, we evaluated the binding of GG to activated peripheral blood mononuclear cells (PBMCs) from cynomolgus macaque and baboon against human. Interestingly, GG only recognized human CD137, while a commercial anti-CD137 mAb (4B4-1), recognized activated PBMCs from both human and non-human primates (NHP). Subsequent analysis revealed that the amino acid sequence of CD137 is largely conserved between primate species ( approximately 95% identical), with the extracellular domain differing by only 9-10 amino acids. Based on these data, we generated mutant constructs in the extracellular domain, replacing NHP with human CD137 sequences, and identified 3 amino acids critical for GG binding. These residues are likely part of a conformational epitope, as a peptide spanning this region is unable to block mAb binding. These data demonstrate that subtle sequence variations of defined co-stimulatory molecules amongst primate species can be employed as a strategy for mapping residues necessary for antibody binding to conformational epitopes.

Background— The Na-Ca exchanger (NCX) is a critical calcium efflux pathway in excitable cells, but little is known regarding its autonomic regulation. Methods and Results— We investigated β-adrenergic receptor and muscarinic receptor regulation of the cardiac NCX in control and heart failure (HF) conditions in atrially paced pigs. NCX current in myocytes from control swine hearts was significantly increased by isoproterenol, and this response was reversed by concurrent muscarinic receptor stimulation with the addition of carbachol, demonstrating “accentuated antagonism.” Okadaic acid eliminated the inhibitory effect of carbachol on isoproterenol-stimulated NCX current, indicating that muscarinic receptor regulation operates via protein phosphatase–induced dephosphorylation. However, in myocytes from atrially paced tachycardia-induced HF pigs, the NCX current was significantly larger at baseline but less responsive to isoproterenol compared with controls, whereas carbachol failed to inhibit isoproterenol-stimulated NCX current, and 8-Br-cGMP did not restore muscarinic responsiveness. Protein phosphatase type 1 dialysis significantly reduced NCX current in failing but not control cells, consistent with NCX hyperphosphorylation in HF. Protein phosphatase type 1 levels associated with NCX were significantly depressed in HF pigs compared with control, and total phosphatase activity associated with NCX was significantly decreased. Conclusions— We conclude that the NCX is autonomically modulated, but HF reduces the level and activity of associated phosphatases; defective dephosphorylation then “locks” the exchanger in a highly active state.

1The transcript of the Na+-Ca2+ exchanger gene NCX1 undergoes alternative splicing to produce tissue-specific isoforms. The cloned NCX1 isoforms were expressed in Xenopus oocytes and studied using a two-electrode voltage clamp method to measure Na+-Ca2+ exchanger activity. 2The cardiac isoform (NCX1.1) expressed in oocytes was less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform (NCX1.3). 3The cardiac isoform of NCX1 is more sensitive to activation by protein kinase A (PKA) than the renal isoform which may be explained by preferential phosphorylation. The cardiac isoform of NCX1 is phosphorylated to a greater extent than the renal isoform. 4The action of PKA phosphorylation which increases the activity of the cardiac isoform of the Na+-Ca2+ exchanger in oocytes was confirmed in adult rat ventricular cardiomyocytes by measuring Na+-dependent Ca2+ flux. 5We conclude that alternative splicing of NCX1 confers distinct functional characteristics to tissue-specific isoforms of the Na+-Ca2+ exchanger.

We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.

A sensitive, highly resolvable, and quantitative method was designed to analyse the diversity of polymerase chain reaction (PCR)-amplified transcripts which possess length polymorphism. A reverse transcriptase-PCR technique was used to amplify rearranged T cell antigen receptor (TCR) transcripts isolated from human blood. Oligonucleotide primers specific for conserved TCR V and C region sequences were used in PCR, with one of the primers end-labeled with 32 P. Amplified cDNA products were analyzed by polyacrylamide sequencing gel electrophoresis with an M13mp18 sequencing ladder as a size marker. 32 P-labeled products were detected by either autoradiography or PhosphorImager. The method allowed determination of the sizes of PCR products with the precision of one nucleotide. The resolution using this technique was much higher than by electrophoresis in agarose gel with ethidium bromide staining. The sizes of PCR products determined by sequencing gel electrophoresis were consistent with the lengths of nucleotide sequences obtained after subcloning PCR products in competent bacterial cells. Analysis of PCR products by sequencing gel electrophoresis was more rapid and as accurate as nucleotide sequence analysis in determining the relative ratios of TCR mRNA in mixtures of T cell clones. The method is applicable for analysis of both rearranged TCR and immunoglobulin genes.

The gene for H-2K class I major histocompatibility antigen on the bm8 variant was cloned and the DNA sequence compared with the parental gene. Sequence analysis demonstrated that seven nucleotides were changed with respect to the parental gene sequence spanning 24 nucleotides. These changes represent an alteration of four amino acids from the parent protein. As this mutation occurred in a single generation, a potential donor gene for such a complex mutation was suggested and identified. The Q4 gene class I-like molecule has a stretch of 95 nucleotides of identity in the region of the bm8 mutation. Genomic Southern analysis of the mutant and parental DNA with a gene-specific oligonucleotide demonstrated that the potential donor gene Q4 is a likely candidate sequence for such an event. The amino acid alterations for the H-2K bm8 mutation are discussed in consideration of the three-dimensional structure of the characterized human class I glycoprotein.

The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by β-adrenergic receptor (β-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni 2+ -sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF ( P P P

Phosphocholine (PC) is the immunodominant epitope found on the surface ofStreptococcus pneumoniae(SPn). T15-idiotype Abs, whose heavy (H) chain variable region is encoded by theV1gene, are dominant in the anti-PC response in adult mice and protect mice from lethal pneumococcal infection. The ability of anti-PC Abs using H chains other than theV1H chain to protect against pneumococcal infection remains controversial. We generated V1−/−knockout mice to determine whether protective anti-PC Abs could be produced in the absence of theV1gene. No anti-PC Abs were produced in V1−/−mice immunized with avirulentSPn; however, PC-BSA binding Abs were induced after immunization with PC-keyhole limpet hemocyanin but at significantly lower levels than those in wild-type mice. These Abs provided poor protection against virulentSPn; thus, −/−mice survived challenge with 104bacteria as compared with 100% survival of V1+/+mice. The anti-PC Abs in V1−/−mice were heteroclitic, binding to nitrophenyl-PC better than to PC. None of nine hybridomas produced from V1−/−mice provided passive protection. However, the V1−/−mice produced normal amounts of Ab toSPnproteins that can partially protect mice againstSPn. These data indicate that theV1gene is critical for the production of anti-PC Abs providing optimum protection against infection withSPn, and the V1−/−mice could be useful in unmasking epitopes other than the immunodominant PC epitope onSPncapable of providing cross protection.

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.

The Na+/Ca2+ exchanger was first identified nearly three decades ago in heart [1] and in nerves [2]. It has been identified in virtually every tissue by functional tests or by molecular or immunological techniques [3–12]. The Na+/Ca2+ exchanger is located in the plasma membrane and serves to extrude calcium from the cytosolic compartment. Nevertheless, it can transport ions in either direction. The direction of transport depends on the electrochemical gradient for sodium and calcium ions across the plasmalemmal membrane since the Na+/Ca2+ exchanger uses the electrochemical gradients to power its transport activity. It transports three sodium ions in one direction and counter-transports one calcium ion (in the opposite direction). Consequently, a net positive charge moves in the direction of sodium transport and transport of sodium and calcium by the Na+/Ca2+ exchanger is both voltage-sensitive and electrogenic [3,4,13].

To the Editor : We read the report of Lin et al. ([1][1]) with great interest. The authors suggest that observed increases in the Ni2+-sensitive bidirectional current after exposure of the mouse, rat, and guinea pig myocytes to isoproterenol were in fact not attributable to changes in the Na+/Ca2+

The gene for the H-2K class I antigen of the bm1 variant was cloned and analyzed at the DNA level and compared with the previously cloned parent B6/Kh gene. Sequence determination and comparative restriction endonuclease studies indicate that Kbm1 is derived from the Kb gene. Seven nucleotide changes within a 13-nucleotide stretch distinguish the mutant from the parent gene and result in amino acid differences at positions 152, 155, and 156 in the antigen. The data confirm previously reported changes at amino acid positions 155 and 156 (arginine to tyrosine and leucine to tyrosine, respectively) and extend the altered region to include two nucleotides encoding a glutamate to alanine substitution at amino acid 152, a change not detected by the protein studies because of limitations of the methods used. The DNA sequence encoding this region of the Kbm1 glycoprotein is identical to the DNA sequence of at least one other known class I gene in the mouse, a finding consistent with the hypothesis that the mutation was not a random event but may be the result of a block transfer of information by a copy mechanism analogous to gene conversion. As the sequence analysis of the coding region for the first 273 amino acid residues shows identity between parent and mutant except for the seven nucleotide changes, all variant-parent functional differences must depend only on the cluster of three amino acid differences in the second domain of the Kb glycoprotein.

The time of replication during the S phase in a murine erythroleukemia (MEL) cell line was determined for immunoglobulin heavy chain constant region C alpha, C gamma 2b and C mu sequences whose boundaries are defined by EcoR1 restriction endonuclease sites (EcoR1 segments). Logarithmically growing cultures of MEL cells with an S phase of about 7.5 hours were pulse labelled with 20 micrograms/ml of 5-bromodeoxyuridine (BUdR). The cells were then fractionated by centrifugal elutriation into 10-12 distinct populations containing cells in different stages of the cell cycle. Flow microfluorimetric (FMF) analysis of DNA content, measurements of cell volume and autoradiography after 3H-thymidine pulse labelling were used to determine position in the cell cycle. Fractions were pooled to represent four selected intervals of S in which BU-DNA was synthesized for 2.5 hrs or less. Newly replicated DNA which had incorporated BUdR into one strand was isolated, cleaved with EcoR1, and separated on neutral Cs2S04 gradients. Equal amounts of BU-DNA replicated during these four intervals of S were electrophoresed in 0.8% agarose gels, transferred to diazotized aminobenzyloxymethyl paper and hybridized with 32p probes containing the C alpha, C gamma 2b and C mu genes and flanking sequences. The relative amounts of segments replicated were assessed by quantitation of the appropriate bands on the autoradiograms by microdensitometry. The results indicate that the 2.8 kb C alpha, 6.6 kb C gamma 2b and 12 kb C mu EcoR1 segments in these MEL cells replicated during defined intervals of the first half of the S phase. The order of replication of these EcoR1 segments as the cells proceeded through S was C alpha, C gamma 2b, C mu, corresponding to the linear order of the genes determined by restriction endonuclease mapping.

In many cells including cardiac myocytes, cytoplasmic Ca is importantly controlled by the plasmalemmal Na-Ca exchanger (3, 8). The tissue diversity and differences in cellular environment raise the question whether the same exchanger is found in all tissues. Recent experiments using rod cells have demonstrated that at least two forms of Na-dependent Ca transport exist. We have examined this issue in various rat and human tissues using the cloned human cardiac Na-Ca exchanger cDNA. Northern blot analysis in these two species show that the major transcript of the Na-Ca exchanger is 7.2 kilobases in heart, brain, kidney, liver, pancreas, skeletal muscle, placenta, and lung. Furthermore, ribonuclease protection analysis in rats shows conservation of the 348-base pair segment tested in heart, brain, kidney, skeletal muscle, and liver. Additionally, Southern blot analysis suggests that a single gene encodes this Na-Ca exchanger. Finally, we show that the clone used to generate our probes encodes a completely functional Na-Ca exchanger. With the use of COS cells and 293 cells transfected with the cloned human cardiac Na-Ca exchanger, we tested the Ca transport properties of the Na-Ca exchanger, the voltage dependence of the Na-Ca exchanger, as well as the Na dependence of the transport function of the Na-Ca exchanger. We conclude that the cardiac form of the Na-Ca exchanger is completely functional when the cDNA is expressed in mammalian cell lines, and, furthermore, this "cardiac" form of the Na-Ca exchanger is naturally expressed in all human and rat tissues tested (but at varying levels).

The mechanism of Ni2+block of the Na+/Ca2+exchanger was examined in Sf 9 cells expressing the human heart Na+/Ca2+exchanger (NCX1-NACA1). As predicted from the reported actions of Ni2+, its application reduced extracellular Na+-dependent changes in intracellular Ca2+concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured63Ni2+entry. The63Ni2+entry was observed in Sf 9 cells expressing the Na+/Ca2+exchanger but not in control cells. The established sequential transport mechanism of the Na+/Ca2+exchanger could be compatible with these results if one of the two ion translocation steps is blocked by Ni2+and the other permits Ni2+translocation. We conclude that, because Ni2+entry was inhibited by extracellular Ca2+and enhanced by extracellular Na+, the Ca2+translocation step moved Ni2+, whereas the Na+translocation step was inhibited by Ni2+. A model is presented to discuss these findings.