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Members of the [Na.sup.2+]/[Ca.sup.2+] exchanger (NCX) family are important regulators of cytosolic [Ca.sup.2+] 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.sup.+]-dependent inactivation and requires much higher levels of regulatory [Ca.sup.2+] to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from [Na.sup.+]-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the [Na.sup.+]-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 [micro]M 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. calcium homeostatis; [Na.sup.+]/[Ca.sup.2+] exchanger; development

The activity of the cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for [Na.sup.+] and [Ca.sup.2+]. In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by [Na.sup.+] and [Ca.sup.2+]. 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.sup.+] and [Ca.sup.+] ([Na.sup.x.sub.i] and [Ca.sup.2+.sub.i]). [Na.sup.+]/[Ca.sup.2+] exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of [Na.sup.+]- and [Ca.sup.2+]-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.sup.+]/[Ca.sup.2+] 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 [Ca.sup.2+], or exposure of patches to limited proteolysis by a-chymotrypsin, reveal that this 'coupling' is due to [Na.sup.+]-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by [Na.sup.+.sub.i]-dependent inactivation was not apparent. The current approach provides a novel means for assessment of [Na.sup.+]/[Ca.sup.2+] exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions. sodium/calcium exchange; coupled inactivation; ionic regulation; giant excised patch

The cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30[degrees]C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the N[H.sub.2]-terminal region of the molecule. Specifically, we found that ~75% of the [Na.sup.+]/[Ca.sup.2+] exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site. myocardial contractility; excitation-contraction coupling; salmonid; calcium handling

The [Na.sup.+]/[Ca.sup.2+] exchanger plays a prominent role in regulating intracellular [Ca.sup.2+] levels in cardiac myocytes and can serve as both a [Ca.sup.2+] influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e., [Ca.sup.2+] entry) of [Na.sup.+]/[Ca.sup.2+] exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on [Na.sup.+]/[Ca.sup.2+] exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac [Na.sup.+]/[Ca.sup.2+] exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) [Na.sup.+]/[Ca.sup.2+] 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 [micro]M 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 [Ca.sup.2+] entry via reverse [Na.sup.+]/[Ca.sup.2+] exchange in this process. sodium/calcium; NCX1.1; giant excised patch clamp; electrophysiology

The cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX), an important regulator of cytosolic [Ca.sup.2+] concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7 [degrees] C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents ([I.sub.NCX]) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation, cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. [I.sub.NCX] of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30 [degrees] C using the giant excised patch technique. The [I.sub.NCX] for both isoforms exhibited [Na.sup.+]-dependent inactivation and [Ca.sup.2+]-dependent positive regulation. The [I.sub.NCX] of NCX1.1 exhibited typical mammalian temperature sensitivities with [Q.sub.10] values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the [I.sub.NCX] of NCX-TR1.0 was relatively temperature insensitive with [Q.sub.10] values of 1.2 and 1.1 for peak and steady-state currents, respectively. [I.sub.NCX] current decay was fit with a single exponential, and the resultant rate constant of inactivation ([Lambda]) was determined as a function of temperature. As expected, [Lambda] decreased monotonically with temperature for both isoforms. Although [Lambda] was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on [Lambda] was not different between the two isoforms. These data suggest that the disparities in [I.sub.NCX] temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after [Alpha]-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in [I.sub.NCX] temperature dependence between the two isoforms are not due to potential disparities in either the [I.sub.NCX] regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments. teleosts; myocardium; contractility; calcium ions

Maxwell, Krista, Jason Scott, Alexander Omelchenko, Anton Lukas, Liyan Lu, Yujuan Lu, Mark Hnatowich, Kenneth D. Philipson, and Larry V. Hryshko. Functional role of ionic regulation of [Na.sup.+]/[Ca.sup.2+] exchange assessed in transgenic mouse hearts. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H2212-H2221, 1999.--[Na.sup.+]/[Ca.sup.2+] exchange is the primary mechanism mediating [Ca.sup.2+] efflux from cardiac myocytes during diastole and, thus, can prominently influence contractile force. In addition to transporting [Na.sup.+] and [Ca.sup.2+], 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 (A680-685), devoid of intracellular [Na.sup.+] ([Na.sup.+])- and [Ca.sup.2+] ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII])-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 [Delta]680-685, 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.sup.+]/[Ca.sup.2+] exchanger, we examined postrest force development in papillary muscles from NCX1.1 and [Delta]680-685 transgenic mice. Postrest potentiation was found to be substantially greater in [Delta]680-685 than in NCX1.1 transgenic mice, supporting the notion that ionic regulation of [Na.sup.+]/[Ca.sup.2+] exchange plays a significant functional role in cardiac contractile properties. giant excised patch; sodium-calcium exchange; ionic regulation; postrest potentiation

Characterizes the regions of NA[sup +/CA[sup 2+] exchanger (NCX) molecule. Sensitivity of trout NCX to low temperature; Localization of the temperature dependence of NCX; Role of NCX in the regulation of Ca[sup 2+] concentration in the cytosol.

Presents a study which evaluated the inhibitory effects of KB-R7943 on sodium[sup+]/calcium[sup2+] exchange currents using the giant excised patch-clamp technique. Methods; Results; Discussion.

Studies the temperature dependence of cloned mammalian and salmonid cardiac Na[sup +]/Ca[sup 2+] exchanger (NCX) isoforms. Exchange currents of NCX of the wild-type trout; Effect of temperature on exchange activity.