Biomedical subjects

K D Philipson

Publications and source records attributed to K D Philipson.

At least 37 records · Page 2Linked to original sources

PubMed1999

Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na(+)-Ca(2+) exchanger.

Ion transport and regulation of Na(+)-Ca(2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(+)-Ca(2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(+)-Ca(2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(+)(i)- (i.e., I(1)) and Ca(2+)(i)- (i.e., I(2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(1) and I(2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with alpha-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(2+)(i), NCX1.3 showed a prominent decrease at higher concentrations (>1 microM). This does not appear to be due solely to competition between Ca(2+)(i) and Na(+)(i) at the transport site, as the Ca(2+)(i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(2+)(i) had only modest effects on Na(+)(i)-dependent inactivation of NCX1.3, whereas I(1) inactivation of NCX1.4 could be completely eliminated by Ca(2+)(i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(+)-Ca(2+) exchange to fulfill tissue-specific requirements of Ca(2+) homeostasis.

Alternative Splicing↗

PubMed1999

Cloning, expression, and characterization of the trout cardiac Na(+)/Ca(2+) exchanger.

Isoform 1 of the cardiac Na(+)/Ca(2+) exchanger (NCX1) is an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7 degrees C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout (Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca(2+) binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at approximately 23 degrees C demonstrated Na(+)-dependent inactivation and Ca(2+)-dependent activation in a manner qualitatively similar to that for NCX1 currents.

Amino Acid Sequence↗

PubMed1999

Cardiac expression of the Na(+)/Ca(2+) exchanger NCX1 is GATA factor dependent.

The cardiac sarcolemmal Na(+)/Ca(2+) exchanger plays a primary role in Ca(2+) efflux and is important in regulating intracellular Ca(2+) and beat-to-beat contractility. Of the three Na(+)/Ca(2+) exchanger genes cloned (NCX1, NCX2, and NCX3), only NCX1 is expressed in cardiac myocytes. NCX1 has alternative promoters for heart, kidney, and brain tissue-specific transcripts. Analysis of the cardiac NCX1 promoter (at -336 bp) identified a cardiac-specific minimum promoter (at -137) and two GATA sites (at -75 and -145). In this study, gel shift and supershift analyses identified GATA-4 in primary neonatal cardiac myocytes. Site-directed mutagenesis of the GATA-4 site at -75 abolishes binding and reduces activity of the minimum and full-length promoters by >90 and approximately 60%, respectively. Mutation of the GATA site at -145 reduces activity of the full-length promoter by approximately 30%. Mutation of an E-box at -175 does not alter promoter activity.

Animals↗

PubMed1999

Functional role of ionic regulation of Na+/Ca2+ exchange assessed in transgenic mouse hearts.

Na+/Ca2+ exchange is the primary mechanism mediating Ca2+ efflux from cardiac myocytes during diastole and, thus, can prominently influence contractile force. In addition to transporting Na+ and Ca2+, the exchanger is also regulated by these ions. Although structure-function studies have identified protein regions of the exchanger subserving these regulatory processes, their physiological importance is unknown. In this study, we examined the electrophysiological and mechanical consequences of cardiospecific overexpression of the canine cardiac exchanger NCX1.1 and a deletion mutant of NCX1.1 (Delta680-685), devoid of intracellular Na+ (Na+i)- and Ca2+ (Ca2+i)- dependent regulatory properties, in transgenic mice. Using the giant excised patch-clamp technique, normal ionic regulation was observed in membrane patches from cardiomyocytes isolated from control and transgenic mice overexpressing NCX1.1. In contrast, ionic regulation was nearly abolished in mice overexpressing Delta680-685, indicating that the native regulatory processes could be overwhelmed by expression of the transgene. To address the physiological consequences of ionic regulation of the Na+/Ca2+ exchanger, we examined postrest force development in papillary muscles from NCX1.1 and Delta680-685 transgenic mice. Postrest potentiation was found to be substantially greater in Delta680-685 than in NCX1.1 transgenic mice, supporting the notion that ionic regulation of Na+/Ca2+ exchange plays a significant functional role in cardiac contractile properties.

Animals↗

PubMed1998

Na+-Ca2+ exchange and sarcoplasmic reticular Ca2+ regulation in ventricular myocytes from transgenic mice overexpressing the Na+-Ca2+ exchanger.

1. The contribution of the sarcoplasmic reticulum (SR) and Na+-Ca2+ exchanger to intracellular Ca2+ regulation in mouse cardiac myocytes was investigated by measuring contraction after variable rest intervals, rapid cooling contractures (RCCs) and fast application of caffeine. The results obtained showed differences from other species in the roles played by the SR and the Na+-Ca2+ exchanger. They suggest that in mouse ventricular myocytes there is significant Ca2+ entry via the exchanger during rest and during the latter part of the Ca2+ transient. 2. In cardiac myocytes isolated from transgenic mice overexpressing the cardiac Na+-Ca2+ exchanger the time to peak and relaxation of twitches and RCCs were faster than in control littermates. The decline of Ca2+, assessed by indo-1 fluorescence, was faster in transgenic myocytes even in the absence of Na+ and Ca2+ in the superfusing solution. This suggests that SR Ca2+ uptake is faster in these myocytes. However, no difference in the expression of SERCA2a, phospholamban or calsequestrin measured with Western blotting could be found in the two groups. 3. We measured SR Ca2+ content by integrating the caffeine-induced transient inward current. The amount of Ca2+ stored in the SR of transgenic mouse myocytes was 69 % greater than in non-transgenic littermates. The increased SR Ca2+ content may be responsible for the faster rate of SR Ca2+ release and uptake in cells from transgenic mice. 4. We performed experiments to assess whether the reversal potential of the Na+-Ca2+ exchanger (ENa-Ca) was different in transgenic cardiac cells. We measured a Ni2+-sensitive current elicited by voltage ramps in non-dialysed myocytes. The current-voltage relationship showed no difference in the reversal potential of the Na+-Ca2+ exchanger in transgenic and control myocytes. This suggests that the effects on the SR Ca2+ content in transgenic cardiac myocytes can be ascribed to the overexpression of the exchanger and are not secondary to changes in intracellular diastolic Ca2+ and Na+.

Animals↗

PubMed1998

Overexpression of the Na(+)-Ca2+ exchanger leads to enhanced inotropic responsiveness to Na(+)-channel agonist without sarcoplasmic reticulum protein changes in transgenic mice.

The present study investigated the influence of over-expression of the cardiac Na(+)-Ca2+ exchanger on myocardial force of contraction and alterations in sarcoplasmic reticulum (SR) Ca(2+)-handling proteins in a transgenic mouse model. Inotropic effects of Na+ channel agonist BDF 9148 and isoprenaline were determined in isolated electrically driven atria. Protein levels of key SR Ca(2+)-handling proteins were determined by Western blot analysis. Transgenic animals had no myocardial hypertrophy or failure. The positive inotropic effect of BDF 9148 was significantly more pronounced in myocardium for transgenic animals, whereas the inotropic response to isoprenaline was similar in both groups. Strong immunoreactivity of the transgene Na(+)-Ca2+ exchanger was detected in myocardium of transgenic animals. Protein levels of SR Ca(2+)-ATPase, phospholamban, and calsequestrin were unchanged. In conclusion, transgenic overexpression of the Na(+)-Ca2+ exchanger is accompanied by increased force development following Na+ channel agonist administration, even though Ca2+ proteins of the SR are unchanged.

Animals↗

PubMed1998

Effects of overexpression of the Na+-Ca2+ exchanger on [Ca2+]i transients in murine ventricular myocytes.

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.

Action Potentials↗

PubMed1998

Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger.

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.

Animals↗

PubMed1998

Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster.

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+o exchanges for bath Na+i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+i affinities and current-voltage relationships, significant differences were observed in their Na+i- and Ca2+i-dependent regulatory properties. Both isoforms underwent Na+i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+i, but their responses to regulatory Ca2+i differed markedly. For both isoforms, the application of cytoplasmic Ca2+i led to a decrease in outward exchange currents. This negative regulation by Ca2+i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+i inhibited peak and steady state currents almost equally, with the extent of inhibition being approximately 80%. In comparison, the effects of regulatory Ca2+i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced ( approximately 20-40% inhibition). For both exchangers, the effects of regulatory Ca2+i occurred by a direct mechanism and indirectly through effects on Na+i-induced inactivation. Our results show that regulatory Ca2+i decreases Na+i-induced inactivation of CALX1.2, whereas it stabilizes the Na+i-induced inactive state of CALX1.1. These effects of Ca2+i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.

Alternative Splicing↗

PubMed1998

Cloning, expression, and characterization of the squid Na+-Ca2+ exchanger (NCX-SQ1).

We have cloned the squid neuronal Na+-Ca2+ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na+-Ca2+ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca2+ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na+-dependent 45Ca2+ uptake; the uptake was inhibited by injection of Ca2+ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na+-dependent inactivation, secondary activation by cytoplasmic Ca2+, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATP gamma S, in the presence of F- (0.2 mM) and vanadate (50 microM), and both effects reversed on application of a phosphatidylinositol-4',5'-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATP gamma S. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4',5'-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5-10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na+ and Ca2+ transport reactions. Outward current transients associated with Na+ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca2+ extrusion were much larger. For NCX-SQ1, charge movements of Ca2+ transport could be defined in voltage jump experiments with a low cytoplasmic Ca2+ (2 microM) in the presence of high extracellular Ca2+ (4 mM). The rates of charge movements showed "U"-shaped dependence on voltage, and the slopes of both charge-voltage and rate-voltage relations (1,600 s-1 at 0 mV) indicated an apparent valency of -0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.

Amino Acid Sequence↗

PubMed1998

Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3).

Three distinct mammalian Na+/Ca2+ exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken a detailed functional comparison of these three exchangers. Each exchanger was stably expressed at high levels in the plasma membranes of BHK cells. Na+/Ca2+ exchange activity was assessed using three different complementary techniques: Na+ gradient-dependent 45Ca2+ uptake into intact cells, Na+ gradient-dependent 45Ca2+ uptake into membrane vesicles isolated from the transfected cells, and exchange currents measured using giant patches of excised cell membrane. Apparent affinities for the transported ions Na+ and Ca2+ were markedly similar for the three exchangers at both membrane surfaces. Likewise, generally similar responses to changes in pH, chymotrypsin treatment, and application of various inhibitors were obtained. Depletion of cellular ATP inhibited NCX1 and NCX2 but did not affect the activity of NCX3. Exchange activities of NCX1 and NCX3 were modestly increased by agents that activate protein kinases A and C. All exchangers were regulated by intracellular Ca2+. NCX1-induced exchange currents were especially large in excised patches and, like the native myocardial exchanger, were stimulated by ATP. Results may be influenced by our choice of expression system and specific splice variants, but, overall, the three exchangers appear to have very similar properties.

Animals↗

PubMed1998

Alternative promoters and cardiac muscle cell-specific expression of the Na+/Ca2+ exchanger gene.

Many studies have investigated the regulation of the Na+/ Ca2+ exchanger, NCX1, but limited data exist on transcriptional regulation of the NCX1 gene. We have identified the transcription start sites of three tissue-specific alternative promoters of NCX1 transcripts from rat heart, kidney, and brain. We have characterized the cardiac NCX1 promoter, from which the most abundant quantities of NCX1 transcripts are expressed. Transfection of primary cardiac myocytes, CHO cells, and COS-7 cells with overlapping genomic DNA fragments spanning the NCX1 cardiac transcription start site has uncovered a cardiac cell-specific minimum promoter from -137 to +85. The cardiac NCX1 promoter is TATA-less but has putative binding sites for cardiac-specific GATA factors, an E box, and an Inr as well as multiple active enhancers. The kidney NCX1 promoter has a typical TATA box and binding sites for several tissue-specific factors. The brain NCX1 promoter is very GC-rich and possesses several Sp-1 binding sites consistent with its ubiquitous expression.

Alternative Splicing↗

PubMed1997

Identification of important amino acid residues of the Na+-Ca2+ exchanger inhibitory peptide, XIP.

The Na+-Ca2+ exchanger plays an important role in cardiac contractility by moving Ca2+ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects of modified peptides on Na+-Ca2+ exchange activity were determined. Exchange activity was assessed as 45Ca2+ uptake into Na+-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule, is required for optimal inhibitory function.

Amino Acid Sequence↗

PubMed1997

Sodium-calcium exchange: recent advances.

Na-Ca exchange proteins are involved in Ca homeostasis in a wide variety of tissues. Unique Na-Ca exchangers have been identified by molecular biological approaches and it appears that these may represent a superfamily of ion transporters, similar to that identified for ion channels. Major advances in our understanding of these transporters have occurred in the past decade by combining molecular approaches with electrophysiological analyses. The regulatory and transport properties of Na-Ca exchangers are beginning to become understood in molecular detail. It also appears that the physiological roles of Na-Ca exchange may be quite complex. This brief review highlights some recent advances in Na-Ca exchange research obtained through the combination of molecular biological and electrophysiological approaches.

Animals↗

PubMed1997

Regulation of cardiac Na(+)-Ca2+ exchanger by the endogenous XIP region.

The cardiac sarcolemmal Na(+)-Ca2+ exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson. 1991. J. Biol. Chem. 266:1014-1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na(+)-dependent inactivation. This inactivation is manifested as a partial decay in outward Na(+)-Ca2+ exchange current after application of Na+ to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na+ affinities. Regulation of the exchanger by nontransported, intracellular Ca2+ was also modified by the XIP-region mutations. Binding of Ca2+ to the intracellular loop activates exchange activity and also decreases Na(+)-dependent inactivation. XIP-region mutants were all still regulated by Ca2+. However, the apparent affinity of the group 1 mutants for regulatory Ca2+ was decreased. The responses of all mutant exchangers to Ca2+ application or removal were markedly accelerated. Na(+)-dependent inactivation and regulation by Ca2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na(+)-induced inactivated state, but that the XIP region is also involved in regulation by Ca2+.

Animals↗

PubMed1997

Calcium signaling in transgenic mice overexpressing cardiac Na(+)-Ca2+ exchanger.

We have produced transgenic mice which overexpress cardiac Na(+)-Ca2+ exchange activity. Overexpression has been assessed by Western blot, Northern blot, and immunofluorescence. Functional overexpression was analyzed using membrane vesicles and isolated ventricular myocytes. In whole cell clamped myocytes dialyzed with 0.1-0.2 mM Fura-2, the magnitude of ICa and Ca2+i-transient triggered by ICa or caffeine were not significantly different in transgenic vs. control myocytes. In transgenic myocytes, activation of ICa, however, was followed by a large slowly inactivating transient inward current representing INa-Ca. This current depended on Ca2+ release as it was abolished when sarcoplasmic reticulum (SR) Ca2+ was depleted using thapsigargin. Cai-transients triggered by rapid application of 5 mM caffeine, even though equivalent in control and transgenic myocytes, activated larger INa-Ca (approximately 5 pA/pF at -90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced Ca2+i-transient and INa-Ca was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni2+ was equally effective in blocking INa-Ca in control or transgenic myocytes. In 9 out of 26 transgenic myocytes, but none of the controls, Ca2+ influx via the exchanger measured at +80 mV caused a slow rise in [Ca2+]i triggering rapid release of Ca2+ from the SR, SR Ca2+ release triggered by the exchanger at such potentials was accompanied by activation of transient current in the inward direction. In 2 mM Fura-2-dialyzed transgenic myocytes caffeine-triggered Cai-transients failed to activate INa-Ca even though the kinetics of inactivation of ICa slowed significantly in caffeine-treated myocytes. In 0.1 mM Fura-2-dialyzed transgenic myocytes 100 microM Cd2+ effectively blocked ICa and suppressed Cai-transients at -10 or +50 mV. Our data suggests that in myocytes overexpressing the exchanger, the content of intracellular Ca2+ pools and the signaling of its release by the Ca2+ channel vis-à-vis the Na(+)-Ca2+ exchanger were not significantly altered despite an up to ninefold increase in the exchanger activity. We conclude that the exchanger remains functionally excluded from the Ca2+ microdomains surrounding the DHP/ryanodine receptor complex.

Animals↗

PubMed1997

Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat.

The gene coding for the Na+/Ca2+ exchanger NCX1 is characterized by a cluster of six exons (A, B, C, D, E, and F) coding for a variable region in the COOH terminus of the large intracellular loop of the protein. Alternative splicing of these exons generates multiple tissue-specific variants of NCX1. Using reverse transcriptase-polymerase chain reaction, we analyzed eight previously described and four new splicing isoforms of NCX1 in a wide variety of tissues and cells. Exons A and B are mutually exclusive, as shown in earlier studies, and splicing isoforms containing exon A are preferentially expressed in heart, brain, and skeletal muscle, whereas splicing variants with exon B are found in all rat tissues except heart. The second and third isoforms of the Na+/Ca2+ exchanger, NCX2 and NCX3, show a deletion of 37 amino acids in the intracellular loop corresponding to parts of the variable region of NCX1. We identified three splicing isoforms of NCX3 in brain and skeletal muscle by reverse transcriptase-polymerase chain reaction. These splice variants are generated by including either of two alternative exons equivalent to the NCX1 exon A or B and by including or excluding a sequence equivalent to the NCX1 exon C. We did not detect any alternative splicing of NCX2. We examined selected tissues from neonatal and adult rats and found developmental regulation for NCX1 and NCX3 splicing isoforms in skeletal muscle. Specific isoform patterns were also detected for NCX1 and NCX3 in cultured cortical neurons, astrocytes, and oligodendrocytes. We suggest a new terminology to distinguish the different splice variants of individual NCX isoforms.

Aging↗

PubMed1996

Cloning of a third mammalian Na+-Ca2+ exchanger, NCX3.

NCX3 is the third isoform of a mammalian Na+-Ca2+ exchanger to be cloned. NCX3 was identified from rat brain cDNA by polymerase chain reaction (PCR) using degenerate primers derived from the sequences of two conserved regions of NCX1 and NCX2. The NCX3 PCR product was used to isolate two overlapping clones totalling 4.8 kilobases (kb) from a rat brain cDNA library. The overlapping clones were sequenced and joined at a unique Bsp106I restriction enzyme site to form a full-length cDNA clone. The NCX3 cDNA clone has an open reading frame of 2.8 kb encoding a protein of 927 amino acids. At the amino acid level, NCX3 shares 73% identity with NCX1 and 75% identity with NCX2 and is predicted to share the same membrane topology as NCX1 and NCX2. Following addition of a poly(A)+ tail to the NCX3 clone, exchanger activity could be expressed in Xenopus oocytes. NCX3 was also expressed in the mammalian BHK cell line. NCX3 transcripts are 6 kb in size and are highly restricted to brain and skeletal muscle. Linkage analysis in the mouse indicated that the NCX family of genes is dispersed, since the NCX1, NCX2, and NCX3 genes mapped to mouse chromosomes 17, 7, and 12, respectively.

Amino Acid Sequence↗