Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

In cardiac-specific Na⁺-Ca²⁺ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca²⁺ current (I_Ca), provides a critical mechanism for the maintenance of Ca²⁺ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na⁺-Ca²⁺ exchanger knockout mice: reduced transsarcolemmal Ca²⁺ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I_to) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I_to was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I_to, ECGs from KO mice exhibited shortened QT intervals. Expression of the I_to-generating K⁺ channel subunit Kv4.2 and the K⁺ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased I_to, reduced I_Ca, and reduced NCX current (I_NCX) on the shape and kinetics of the AP. Reduction of I_Ca and elimination of I_NCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I_to was increased in the computer model. Thus, the increase in I_to, and not the reduction of I_Ca or I_NCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I_to may comprise an important regulatory mechanism to limit Ca²⁺ influx via a reduction of AP duration, thus preventing Ca²⁺ overload in situations of reduced myocyte Ca²⁺ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current     

Role of Na+-Ca2+ exchanger in myocardial ischemia/reperfusion injury: evaluation using a heterozygous Na+-Ca2+ exchanger knockout mouse model

We used Na(+)-Ca(2+) exchanger (NCX) knockout mice to evaluate the effects of NCX in cardiac function and the infarct size after ischemia/reperfusion injury. The contractile function in NCX KO mice hearts was significantly better than that in wild type (WT) mice hearts after ischemia/reperfusion and the infarct size was significantly small in NCX KO mice hearts compared with that in WT mice hearts. NCX is critically involved in the development of ischemia/reperfusion-induced myocardial injury and therefore the inhibition of NCX function may contribute to cardioprotection against ischemia/reperfusion injury.     

Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside- out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.     

Immunological crossreactivity between the retinal Na+-Ca2+,K+ and the cardiac Na+-Ca2+ exchanger proteins

We have used a series of monoclonal antibodies (mAbs) to determine the degree of microscopic structural homology between the retinal Na⁺-Ca²⁺, K⁺ and the cardiac Na⁺-Ca²⁺ exchange proteins. Sets of mAbs were raised separately to partially purified preparations of either the retinal or the recombinant myocardial exchanger. Each panel of mAbs was then screened for crossreactivity with the respective heterologous exchanger using enzyme-linked immunoassay and immunoblotting techniques. Out of 43 anti-retinal exchanger mAbs, we found 3 detecting the cardiac exchanger on immunoblots, while 4 out of 36 anti-cardiac exchanger mAbs reacted with the retinal exchanger. The strength of the crossreactions was generally weak and suggested that only low affinity epitopes were available on the heterologous proteins. For two crossreacting anti-retinal mAbs the apparent binding affinities to the cardiac exchanger were lower by more than two orders of magnitude. The overall low degree of epitope sharing among the two sets of mAbs confirms that in spite of their obvious functional and topological similarities, microscopic structural homologies between the two proteins are scarce.     

L型钙流和反向钠-钙交换在豚鼠心室肌细胞兴奋-收缩偶联中的作用

目的 :比较和探讨L型钙流 [ICa(L) ]和反向钠—钙交换 (NCX)在触发豚鼠心室肌细胞兴奋—收缩偶联中的作用。方法 :以分离的豚鼠单个心室肌细胞为对象 ,采用膜片钳和单细胞收缩测量技术 ,给予 35℃的各种含药物细胞外液快速灌流 ,同时记录ICa(L) 和细胞收缩。结果 :①在 +10mV的钳制电压 ,使用硝苯地平 (Nif) 10～ 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L ,阻滞ICa(L) 越多 ,细胞收缩被阻滞得越多 ,呈线性相关。②在 +5 0mV的钳制电压 ,Nif 10 0 μmol/L以及Nif 30 μmol/L +Cd2 +3 0 μmol/L仅能抑制部分细胞收缩 ,但剩余的细胞收缩起始时间明显延迟 ,且能被 5mmol/LNi2 +所阻滞。③在 +10 0mV的钳制电压 ,细胞收缩起始时间较 +5 0mV明显延迟 ,且不能被Nif 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L所阻滞。结论 :在生理条件下 ,ICa(L) 是触发心室肌细胞兴奋—收缩偶联的主要途径 ,但在膜电位 >+5 0mV时 ,反向NCX也参与兴奋—收缩偶联。     

mRNA-induced expression of the cardiac Na+-Ca2+ exchanger in Xenopus oocytes

Xenopus oocytes were injected with total mRNA isolated from hearts of 1-day-old chicks. After 5 days of incubation the follicular cell layers were removed and the oocytes were loaded with Na+ by incubation in hypertonic EGTA solution at 37 degrees C. The Na+-loaded oocytes accumulated 45Ca2+ from a Na+-free medium at a 3-18-fold higher rate than noninjected oocytes or oocytes injected with control solution containing no mRNA. Oocytes not subjected to the Na+-loading procedure showed no mRNA-dependent 45Ca2+ uptake. Size fractionation of the mRNA using sucrose density gradient centrifugation under denaturing conditions led to the identification of a 25 S fraction competent for induction of the Na+-Ca2+ exchange system.     

Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+

The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1- 10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump.

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca(2+) channels was assessed by whole-cell patch-clamp recordings of Ca(2+) current and complementary experiments in which internal [Na(+)] was reduced. However, Ca(2+) entry via Na(+)-Ca(2+) exchange measured in the absence of L-type Ca(2+) current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca(2+) channels increase [Ca(2+)] near Na(+)-Ca(2+) exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca(2+) release in heart cells under various conditions.     

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.

     

Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins alpha-actinin and dystrophin

Theactin-binding proteins dystrophin and -actinin are members of afamily of actin-binding proteins that may link the cytoskeleton tomembrane proteins such as ion channels. Previous work demonstrated thatthe activity of Ca²⁺ channels can be regulated by agentsthat disrupt or stabilize the cytoskeleton. In the present study, weemployed immunohistochemical and electrophysiological techniques toinvestigate the potential regulation of cardiac L-type Ca²⁺channel activity by dystrophin and -actinin in cardiac myocytes andin heterologous cells. Both actin-binding proteins were found tocolocalize with the Ca²⁺ channel in mouse cardiac myocytesand to modulate channel function. Inactivation of the Ca²⁺channel in cardiac myocytes from mice lacking dystrophin(mdx mice) was reduced compared with that in wild-typemyocytes, voltage dependence of activation was shifted by 5 mV to morepositive potentials, and stimulation by the -adrenergic pathway andthe dihydropyridine agonist BAY K 8644 was increased. Furthermore, heterologous coexpression of the Ca²⁺ channel with muscle,but not nonmuscle, forms of -actinin was also found to reduceinactivation. As might be predicted from a reduction ofCa²⁺ channel inactivation, a prolonging of the mouseelectrocardiogram QT was observed in mdx mice. These resultssuggest a combined role for dystrophin and -actinin in regulatingthe activity of the cardiac L-type Ca²⁺ channel and apotential mechanism for cardiac dysfunction in Duchenne and Beckermuscular dystrophies.

     

Three-dimensional distribution of cardiac Na+-Ca2+ exchanger and ryanodine receptor during development

Mechanisms of cardiac excitation-contraction coupling in neonates are still not clearly defined. Previous work in neonates shows reverse-mode Na(+)-Ca(2+) exchange to be the primary route of Ca(2+) entry during systole and the neonatal sarcoplasmic reticulum to have similar capability as that of adult in storing and releasing Ca(2+). We investigated Na(+)-Ca(2+) exchanger (NCX) and ryanodine receptor (RyR) distribution in developing ventricular myocytes using immunofluorescence, confocal microscopy, and digital image analysis. In neonates, both NCX and RyR clusters on the surface of the cell displayed a short longitudinal periodicity of approximately 0.7 microm. However, by adulthood, both proteins were also found in the interior. In the adult, clusters of NCX on the surface of the cell retained the approximately 0.7-microm periodicity whereas clusters of RyR adopted a longer longitudinal periodicity of approximately 2.0 microm. This suggests that neonatal myocytes also have a peri-M-line RyR distribution that is absent in adult myocytes. NCX and RyR colocalized voxel density was maximal in neonates and declined significantly with ontogeny. We conclude in newborns, Ca(2+) influx via NCX could potentially activate the dense network of peripheral Ca(2+) stores via peripheral couplings, evoking Ca(2+)-induced Ca(2+) release.     

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.     

Cooperative interaction between Ca2+ binding sites in the hydrophilic loop of the Na+-Ca2+ exchanger

A high affinity Ca²⁺-binding domain which is located in a middle portion of the large intracellular loop of the Na⁺-Ca²⁺ exchanger contains two highly acidic sequences, each characterized by three consecutive aspartic acid residues (Levitsky DO, Nicoll DA, and Philipson KD (1994) J Biol Chem 269: 22847–22852). This portion of the protein provides secondary Ca²⁺ regulation of the exchanger activity. To determine number of Ca²⁺ binding sites participating in formation of the high affinity domain, we isolated polypeptides of different lengths encompassing the domain and measured ⁴⁵Ca²⁺ binding. The fusion proteins containing the high affinity domain were obtained in a Ca²⁺-bound form and as evidenced by shifts in there mobility in SDS-polyacrylamide gels after EGTA treatment. The Ca²⁺ binding curves obtained after equilibrium dialysis reached saturation at 1 M free Ca²⁺, Kd value being approx. 0.4 M. The Ca²⁺ binding occured in a highly cooperative manner. Upon saturation, the amount of Ca²⁺ ion bound varied from 1.3–2.1 mot per mot protein. Proteins with an aspartate in each acidic sequence mutated lacked the positive cooperativity, had lower Ca²⁺ affinity and bound two to three times less Ca²⁺. Na⁺-Ca²⁺ exchangers of tissues other than heart though different from the cardiac exchanger by molecular weight most likely possess a similar Ca²⁺ binding site. It is concluded that, by analogy with Ca²⁺ binding proteins of EF-type, the high Ca²⁺-affinity domain of the Na⁺-Ca²⁺ exchanger is comprised of at least two binding sites interacting cooperatively.     

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na+-Ca2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca²⁺ and Na⁺. These results led us to believe that the increase in Na⁺ by taurine could be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through Na⁺-Ca²⁺ exchange. Therefore, we investigated the effect of -alanine, a blocker of the taurine-Na⁺ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2,4-dimethylbenzamil), a Na⁺-Ca²⁺ exchanger blocker on taurine-induced [Ca]_i increase in embryonic chick heart cells. Using Fura-2 Ca²⁺ imaging and Fluo-3 Ca²⁺ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]_i at both the cytosolic and the nuclear levels. Preexposure to 500 M of the blocker of the taurine-Na⁺ cotransporter, -alanine, prevented the amino acid-induced increase of total [Ca]_i. On the other hand, application of -alanine did not reverse the action of taurine on total [Ca]_i. However, low concentrations of the Na⁺-Ca²⁺ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of -alanine). Thus, the effect of taurine on [Ca]_i in heart cells appears to be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through the Na⁺-Ca²⁺ exchanger.