Search

μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFβ1as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFβ/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFβ1induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFβ1through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFβ1stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFβ1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

The activity of the cardiac Na+/Ca2+ exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for Na+ and Ca2+. In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by Na+ and Ca2+. In an effort to evaluate how ionic regulation influences exchange activity under pulsatile conditions, we studied the behavior of the cloned NCX1.1 during frequency-controlled changes in intracellular Na+ and Ca+ (Na[Formula: see text] and Ca[Formula: see text]). Na+/Ca2+ exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of Na+- and Ca2+-dependent ionic regulation so that the effects of variables such as pulse frequency and duration could be optimally discerned. We demonstrate that increasing the frequency or duration of solution pulses leads to a progressive decline in pure outward, but not pure inward, Na+/Ca2+ exchange current. However, when the exchanger is permitted to alternate between inward and outward transport modes, both current modes exhibit substantial levels of inactivation. Changes in regulatory Ca2+, or exposure of patches to limited proteolysis by α-chymotrypsin, reveal that this “coupling” is due to Na+-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by Na[Formula: see text]-dependent inactivation was not apparent. The current approach provides a novel means for assessment of Na+/Ca2+ exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions.

SEA0400 (SEA) blocks cardiac and neuronal Na+-Ca2+ exchange with the highest affinity of any known inhibitor, yet very little is known about its molecular mechanism of action. Previous data from our lab suggested that SEA stabilizes or modulates the transition of NCX1.1 exchangers into a Na+i-dependent (I1) inactive state. To test this hypothesis, we examined the effects of SEA on mutant exchangers with altered ionic regulatory properties. With mutants where Na+i-dependent inactivation is absent, the effects of SEA were greatly reduced. Conversely, with mutants displaying accelerated Na+i-dependent inactivation, block of NCX1.1 by SEA was either enhanced or unchanged, depending upon the phenotype of the particular mutation. With mutant exchangers where Ca2+i-dependent (I2) inactivation was suppressed, block of exchange currents by SEA was similar to that observed for wild-type NCX1.1. These data strongly support the involvement of I1 inactivation in the inhibitory mechanism of NCX1.1 by SEA, whereas I2 inactivation does not seem to serve an important role. The involvement of processes regulated by intracellular Na+ in the inhibitory mechanism of SEA may prove to be particularly important when considering the potential cardioprotective effects of this agent.

The electrophysiological effects of the benzothiazepine 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157) (CGP) were investigated on the canine (NCX1.1) and Drosophila (CALX1.1) plasmalemmal Na+-Ca2+ exchangers. These exchangers were selected for study because they show opposite responses to cytoplasmic regulatory Ca2+, thereby allowing us to examine the role of this regulatory mechanism in the inhibitory effects of CGP. CGP blocked Na+-Ca2+ exchange current mediated by both transporters with moderate potency (IC50 values = approximately 3-17 microM) compared with other recently reported blockers of Na+-Ca2+ exchange [e.g., 2-[4-[2,5-difluorophenyl) methoxy]phenoxy]phenoxy]-5-ethoxyaniline (KB-R7943) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (SEA0400)]. Experiments using alpha-chymotrypsin to remove autoregulation of Na+-Ca2+ exchange showed that block by CGP was reduced, suggesting that part of the effects of this drug may require intact ionic regulatory mechanisms. For NCX1.1, the inhibition produced by CGP was greater for outward Na+-Ca2+ exchange currents compared with inward currents. When CALX1.1 was examined, the extent of inhibition was similar for both inward and outward exchange currents. Although the extent and potency of CGP-mediated inhibition of Na+-Ca2+ exchange are less than those observed with SEA0400 and KB-R7943, our data demonstrate that CGP constitutes a novel class of plasmalemmal Na+-Ca2+ exchange inhibitors. Moreover, the widespread use of CGP as a selective mitochondrial Na+-Ca2+ exchange inhibitor should be reconsidered in light of these additional inhibitory effects.

Remodeling of the extracellular matrix is beneficial during the acute wound healing stage following tissue injury. In the short term, resident fibroblasts and myofibroblasts regulate the matrix remodeling process through production of matricellular protein components that provide structural support to the damaged tissue. This process is largely governed by the transforming growth factor-β1(TGF-β1) pathway, a critical mediator of the remodeling process. In the long term, chronic activation of the TGF-β1pathway promotes excessive synthesis and deposition of matrix proteins, including fibrillar collagens, which ultimately leads to organ failure. SnoN (and its alternatively-spliced isoforms SnoN2, SnoA, and SnoI) is one of four members of a family of negative regulators of TGF-β1signaling that includes Ski and functional Smad-suppressing elements on chromosomes 15 and 18. SnoN has been shown to be structurally and functionally similar to Ski and has been demonstrated to directly interact with Ski to abrogate gene expression. Despite this, little progress has been made in delineating a specific role for SnoN in the regulation of myofibroblast phenotype and function. This review outlines the current body of knowledge of what we refer to as the “Ski-Sno superfamily,” with a focus on the structural and functional importance of SnoN in mediating the fibrotic response by myofibroblasts following tissue injury.

The Na+/Ca2+ exchanger plays a prominent role in regulating intracellular Ca2+ levels in cardiac myocytes and can serve as both a Ca2+ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e. , Ca2+ entry) of Na+/Ca2+ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na+/Ca2+exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na+/Ca2+exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na+/Ca2+ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 μM KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca2+ entry via reverse Na+/Ca2+ exchange in this process.

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 (Δ680–685), devoid of intracellular Na+([Formula: see text])- and Ca2+([Formula: see text])- 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 Δ680–685, 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 Δ680–685 transgenic mice. Postrest potentiation was found to be substantially greater in Δ680–685 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.

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.

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.

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+ i- and Ca2+ i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that μm levels of Ca2+ i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+ i binding site of CALX1.1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+ i to inhibit Na+-Ca2+exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Δ310–313) of Na+ i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+ i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+ i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+ i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+ i. This result indicates that the regulatory Ca2+ i binding site of NCX1.1 retains function in a CALX1.1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+ i binding signal.

Studies were directed at determining whether hepatocytes, isolated from female Sprague-Dawley rats, facilitate the uptake of protein-bound long-chain fatty acids. We postulated one form of facilitated uptake may occur through an ionic interaction between the protein-ligand complex and the cell surface. These interactions are expected to supply additional ligand to the cell for uptake. The clearance rate of [3H]-palmitate in the presence of α1-acid-glycoprotein (pI=2.7), albumin (pI=4.9) and lysozyme (pI=11.0) was investigated. Palmitate uptake was determined in the presence of protein concentrations that resulted in similar unbound ligand fractions (=0.03). The experimental clearance rates were compared to the theoretical predictions based upon the diffusion-reaction model. By use of our experimentally determined equilibrium binding and dissociation rate constants for the various protein-palmitate complexes, the diffusion-reaction model predicted clearance rates were 4.9 μl s−1/106 cells, 4.8 μl s−1/106 cells and 5.5 μl s−1/106 cells for α1-acid-glycoprotein, albumin and lysozyme, respectively; whereas the measured hepatocyte palmitate clearance rates were 1.2±0.1 μl s−1/106 cells, 2.3±0.3 μl s−1/106 cells and 7.1±0.7 μl s−1/106, respectively. Hepatocyte palmitate clearance was significantly faster (P

Cardiac muscle fails to relax upon replacement of extracellular Ca 2+ with Ba 2+ . Among the manifold consequences of this intervention, one major possibility is that Na + -Ba 2+ exchange is inadequate to support normal relaxation. This could occur due to reduced transport rates of Na + -Ba 2+ exchange and/or by failure of Ba 2+ to activate the exchanger molecule at the high affinity regulatory Ca 2+ binding site. In this study, we examined transport and regulatory properties for Na + -Ca 2+ and Na + -Ba 2+ exchange. Inward and outward Na + -Ca 2+ or Na + -Ba 2+ exchange currents were examined at 30°C in giant membrane patches excised from Xenopus oocytes expressing the cloned cardiac Na + -Ca 2+ exchanger, NCX1. When excised patches were exposed to either cytoplasmic Ca 2+ or Ba 2+ , robust inward Na + -Ca 2+ exchange currents were observed, whereas Na + -Ba 2+ currents were absent or barely detectable. Similarly, outward currents were greatly reduced when pipette solutions contained Ba 2+ rather than Ca 2+ . However, when solution temperature was elevated from 30°C to 37°C, a substantial increase in outward Na + -Ba 2+ exchange currents was observed, but not so for inward currents. We also compared the relative abilities of Ca 2+ and Ba 2+ to activate outward Na + -Ca 2+ exchange currents at the high affinity regulatory Ca 2+ binding site. While Ba 2+ was capable of activating the exchanger, it did so with a much lower affinity ( K D ∼ 10 μM) compared with Ca 2+ ( K D ∼ 0.3 μM). Moreover, the efficiency of Ba 2+ regulation of Na + -Ca 2+ exchange is also diminished relative to Ca 2+ , supporting ∼60% of maximal currents obtainable with Ca 2+ . Ba 2+ is also much less effective at alleviating Na + i -induced inactivation of NCX1. These results indicate that the reduced ability of NCX1 to adequately exchange Na + and Ba 2+ contributes to failure of the relaxation process in cardiac muscle.

Survival of myocytes and mesenchymal cells in the heart is tightly regulated by a number of adaptive processes that are invoked with the changes that occur within the parenchyma and stroma. Autophagy is implicated in cellular housekeeping duties and maintenance of the integrity of the intracellular milieu by removal of protein aggregates and damaged organelles, whereas under pathophysiological conditions, the chronic up-regulation of autophagy may lead to significant disturbance of homeostatic conditions. Nonetheless, the role of autophagy in heart disease in the context of cardiac ischemia-reperfusion injury is currently unclear. This review will focus upon the role of autophagy as it pertains to ischemia reperfusion damage in the heart.

Bovine serum albumin (albumin) was modified by treatment with nitric oxide (NO) to form S -nitrosoalbumin. Analysis of the reduced sulfhydryl groups showed that more than 99% of the albumin was converted to S -nitrosoalbumin. Using a 1:1 molar ratio of protein:palmitate, the unbound palmitate fraction in the presence of S -nitrosoalbumin was determined to be greater (28%) than in the presence of albumin as determined by heptane:water partitioning. NO degradation products neither affected the palmitate heptane:water partition ratio in the absence of binding protein nor the hepatocyte uptake of [ 3 H]palmitic acid. The equilibrium association constants ( K a ) for albumin-palmitate and S -nitrosoalbumin-palmitate complexes were determined using the stepwise equilibrium model. The K α for the first and second palmitate binding sites were (4.6 ± 1.2) × 10 8 M −1 and (3.3 ± 0.5) × 10 7 M −1 and (3.1 ± 0.9) × 10 8 M −1 and (1.3 ± 0.8) × 10 8 M −1 for albumin and S -nitrosoalbumin, respectively. Thus, the increased unbound fraction of palmitate in the presence of S -nitrosoalbumin was apparently due to a decreased binding affinity at the first high-affinity binding site. Palmitate uptake by hepatocyte suspensions was 27% higher in the presence of S -nitrosoalbumin as compared with albumin. This increase paralleled the increased unbound palmitate fraction. When the albumin concentration was adjusted to account for the increased unbound fraction, there was no difference in the palmitate uptake rates between albumin and S -nitrosoalbumin. Our findings indicate that under conditions where NO concentrations are high (e.g. cirrhosis) and extensive S -nitrosylation of serum albumin occurs, the decreased ligand binding ability of S -nitrosoalbumin may be an important consideration when modeling drug uptake in pathological states.

Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Members of the Na+/Ca2+exchanger (NCX) family are important regulators of cytosolic Ca2+in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene ( NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish ( Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 μM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.

SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has recently been described as a potent and selective inhibitor of Na(+)-Ca(2+) exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of SEA0400 were investigated on the cloned cardiac Na(+)-Ca(2+) exchanger, NCX1.1, expressed in Xenopus laevis oocytes to gain insight into its inhibitory mechanism. Na(+)-Ca(2+) exchange currents were measured using the giant excised patch technique using conditions to evaluate both inward and outward currents. SEA0400 inhibited outward Na(+)-Ca(2+) exchange currents with high affinity (IC(50) = 78 +/- 15 and 23 +/- 4 nM for peak and steady-state currents, respectively). Considerably less inhibitory potency (i.e., micromolar) was observed for inward currents. The inhibitory profile was reexamined after proteolytic treatment of excised patches with alpha-chymotrypsin, a procedure that eliminates ionic regulatory mechanisms. After this treatment, an IC(50) value of 1.2 +/- 0.6 microM was estimated for outward currents, whereas inward currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400 on outward exchange currents were evident at both high and low concentrations of regulatory Ca(2+), although distinct features were noted. SEA0400 accelerated the inactivation rate of outward currents. Based on paired pulse experiments, SEA0400 altered the recovery of exchangers from the Na(+)(i)-dependent inactive state, particularly at higher regulatory Ca(2+)(i) concentrations. Finally, the inhibitory potency of SEA0400 was strongly dependent on the intracellular Na(+) concentration. Our data confirm that SEA0400 is the most potent inhibitor of the cardiac Na(+)-Ca(2+) exchanger described to date and provide a reasonable explanation for its apparent transport mode selectivity.

The Na+-Ca2+ exchanger (NCX) is an integral protein essential for cellular Ca2+ homeostasis. Previous structure-function studies have identified regulatory regions within the exchanger's cytoplasmic domain, of which the XIP region shown to be involved in the rate and extent of Na+-dependent inactivation (I1). Although the XIP region is highly conserved amongst NCX isoforms, distinct I1 phenotypes exist. To better define the role of this region, we constructed chimaeric NCX1:NCX2.1 proteins with their respective XIP regions interchanged as well as amino acid substitutions within the XIP region to examine the more subtle aspects of phenotypic differences between NCX1.3 and NCX2.1. Mutant exchangers were expressed in Xenopus oocytes, and outward Na+-Ca2+ exchange activity was assessed using the giant, excised patch clamp technique. Substitution of the XIP region of NCX1.4 with the corresponding region from NCX2.1 caused an apparent loss of I1 whereas a reduction in the extent of inactivation and a 15-fold increase in the rate of recovery from I1 were observed in the NCX1.3 - XIP2 chimaera. Similarly, substitution of charged amino acids within the XIP region in NCX1.3 caused a slight increase in the rate of recovery, equivalent to that observed for NCX2.1. Thus, non-conserved residues in the XIP region may be essential in maintaining the structural stability of the Na+-dependent inactive state of NCX1. Furthermore, the XIP region must interact with other regulatory domains of the protein, such as the mutually exclusive exon, thereby contributing to the structure-function relationship as well as the regulatory phenotype of each Na+-Ca2+ exchanger variant and isoform.

Radioligand binding and displacement experiments are often interpreted in terms of simple competition between two ligands for occupancy of a single binding site on a receptor. Given our current understanding of the complexities of receptor structure and function, it is probable that more complex interactions occur in many cases. By analysis of a hypothetical two-site receptor model, we show that apparent competitive inhibition can arise in several ways, depending on the specificities of the two sites and the interactions between them. We show that binding experiments can in some cases be used to rule out certain models from among a group of apparently plausible ones, provided that experimental criteria are met which permit a meaningful statistical comparison of models to be made. Ideally, these should include: i) an independent study of ligand and inhibitor binding in the absence of each other; ii) carrying out saturation binding and displacement experiments over as wide a range of ligand and inhibitor concentrations as possible; iii) computerized curve-fitting and statistical analysis as a tool for model-testing. While practical limitations may restrict the attainment of such goals, a thorough study of the equilibrium binding properties of a particular receptor system provides the foundation for the design of more definitive experiments at the molecular level, upon which the proof of any binding model ultimately must rest.

[ 3 H]Etorphine binding was characterized in rat brain homogenates depleted of endogenous opioids from animals acutely and chronically treated with morphine or naloxone and either unstressed or subjected to a 3-h restraint period in the cold. There was significant reduction in the number of high-affinity opiate binding sites in brain tissue from stressed as compared to unstressed animals. Despite the fact that the opiate drug regimens used produce marked behavioral and physiological effects, stress-induced opiate receptor depletion was not influenced by the drugs or by withdrawal. The various drug treatments also failed to produce significant changes in opiate receptor site densities or affinities in either stressed or unstressed animals. We propose that persistent activation of opiate receptors by endogenous opioids released during restraint stress leads to receptor ‘down-regulation’.

Endogenous substances that modulate the activity of (Na+ + K+)-ATPase through interaction at the cardiac glycoside site have been postulated. Reports of digitalis-like biological and immunological activity exhibited by certain ACTH/MSH peptides and 14-OH steroids make these compounds potential candidates as endogenous digitalis-like factors. We tested several ACTH/MSH peptides and 14 alpha-OH steroids in four in vitro assays and detected no significant cardiac glycoside-like activity. On the other hand, chlormadinone acetate, a progesterone derivative shown to bind with high affinity to the digitalis receptor, was nearly equipotent to digoxigenin in a [3H]ouabain radioreceptor assay. In a [3H]digoxin radioimmunoassay, however, digoxigenin and digoxin were equipotent but chlormadinone acetate was inactive. A clear dissociation between radioreceptor assay and radioimmunoassay activity was also observed using 15 beta-OH-progesterone. Our findings indicate that (a) ACTH/MSH peptides and 14 alpha-OH steroids are not viable candidates as endogenous digitalis-like factors, (b) digoxin antibodies are not necessarily directed at molecular determinants critical for biological activity, and (c) among the compounds reported to exhibit digitalis-like activity and postulated to share structural features with an endogenous steroidal digitalis-like factor, only chlormadinone acetate and its congeners appear to constitute tenable models.