Larry V. Hryshko

STRUCTURE-FUNCTION ANALYSIS OF CALX1.1, A NA+-CA2+EXCHANGER FROM DROSOPHILA : MUTAGENESIS OF IONIC REGULATORY SITES

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.

Crystal Structure of CBD2 from the Drosophila Na+/Ca2+ Exchanger: Diversity of Ca2+ Regulation and Its Alternative Splicing Modification

Na(+)/Ca(2+) exchangers (NCXs) promote the extrusion of intracellular Ca(2+) to terminate numerous Ca(2+)-mediated signaling processes. Ca(2+) interaction at two Ca(2+) binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca(2+) regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca(2+) interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca(2+) binding. The carboxylate residues that coordinate two Ca(2+) in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca(2+) regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca(2+) binding site to change Ca(2+) binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca(2+) regulatory behavior.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na+/Ca2+ exchangers (NCX) constitute a major Ca2+ export system that facilitates the re-establishment of cytosolic Ca2+ levels in many tissues. Ca2+ interactions at its Ca2+ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na+/Ca2+ exchange activity. The structure of the Ca2+-bound form of CBD1, the primary Ca2+ sensor from canine NCX1, but not the Ca2+-free form, has been reported, although the molecular mechanism of Ca2+ regulation remains unclear. Here, we report crystal structures for three distinct Ca2+ binding states of CBD1 from CALX, a Na+/Ca2+ exchanger found in Drosophila sensory neurons. The fully Ca2+-bound CALX-CBD1 structure shows that four Ca2+ atoms bind at identical Ca2+ binding sites as those found in NCX1 and that the partial Ca2+ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca2+ binding at CBD1. The structures also predict that the primary Ca2+ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu455, which coordinates the primary Ca2+ pair, produces dramatic reductions of the regulatory Ca2+ affinity for exchange current, whereas mutagenesis of Glu520, which coordinates the secondary Ca2+ pair, has much smaller effects. Furthermore, our structures indicate that Ca2+ binding only enhances the stability of the Ca2+ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca2+ 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.

Extracellular and intracellular proteases in cardiac dysfunction due to ischemia-reperfusion injury

Various procedures such as angioplasty, thrombolytic therapy, coronary bypass surgery, and cardiac transplantation are invariably associated with ischemia-reperfusion (I/R) injury. Impaired recovery of cardiac function due to I/R injury is considered to be a consequence of the occurrence of both oxidative stress and intracellular Ca(2+)-overload in the myocardium. These changes in the ischemic myocardium appear to activate both extracellular and intracellular proteases which are responsible for the cleavage of extracellular matrix and subcellular structures involved in the maintenance of cardiac function. It is thus intended to discuss the actions of I/R injury on several proteases, with a focus on calpain, matrix metalloproteinases, and cathepsins as well as their role in inducing alterations both inside and outside the cardiomyocytes. In addition, modifications of subcellular organelles such as myofibrils, sarcoplasmic reticulum and sarcolemma as well as extracellular matrix, and the potential regulatory effects of endogenous inhibitors on protease activities are identified. Both extracellular and intracellular proteolytic activities appear to be imperative in determining the true extent of I/R injury and their inhibition seems to be of critical importance for improving the recovery of cardiac function. Thus, both extracellular and intracellular proteases may serve as potential targets for the development of cardioprotective interventions for reducing damage to the heart and retarding the development of contractile dysfunction caused by I/R injury.

Control of interval-force relation in canine ventricular myocardium studied with ryanodine

1 The mechanism of post‐extrasystolic, rest and frequency potentiation was studied in canine isolated ventricular muscle. 2 Ryanodine, which impairs Ca availability from the sarcoplasmic reticulum (SR), reduced the amplitude of the extrasystole less than that of the steady state contraction. Ryanodine also inhibited post‐extrasystolic potentiation and converted rest‐potentiation into rest depression. Rest‐potentiation was blocked preferentially by ryanodine compared to post‐extrasystolic potentiation. An increase in the contribution of extracellular Ca to the extrasystolic contraction could not entirely account for the post‐extrasystolic potentiation. 3 Prolonged rest, by itself, also caused depression of the first post‐rest contraction. During rest‐potentiation, SR Ca seemed to play a greater role in contraction than transmembrane Ca influx. However, the ability of the ‘release pool’ of Ca in the SR to be reprimed after a contraction was reduced. This was seen as a decrease in post‐extrasystolic potentiation elicited immediately after rest. 4 A decrease in stimulus interval was associated with a transient decrease in contraction amplitude followed by an increase. An abrupt increase in stimulus interval had the opposite effect. Ryanodine blocked the initial transient changes and accelerated the delayed changes. These results suggest that the transient changes in contraction after sudden changes in drive interval are dependent on the SR. 5 Transmembrane Ca entry and the rate of recovery of the Ca release process (repriming) in the SR after a contraction seem to be interval‐dependent. The data also indicate that different mechanisms are involved in post‐extrasystolic and rest‐potentiation. 6 The results are consistent with a model which proposes ‘recirculation’ of activator Ca within the SR after a contraction or of the presence of an appreciable amount of inactivation of the SR Ca release process during normal stimulation. An increased pool of releasable Ca due to longer recirculation time or a time‐dependent decay in the level of inactivation of Ca release from the SR may give rise to rest‐potentiation.

Effects of caffeine and ryanodine on depression of post‐rest tension development produced by Bay K 8644 in canine ventricular muscle

1 Post‐rest inotropy in canine ventricular myocardium has proved a useful indicator of sarcoplasmic reticular calcium release. This phenomenon is converted to rest depression by the calcium channel activator (agonist), Bay K 8644 as well as other chemically diverse agents such as caffeine and ryanodine. 2 Rapid cooling contractures and post‐rest contraction amplitude were used as independent measures of sarcoplasmic reticular calcium content and release. Simultaneous recordings of transmembrane action potentials and their accompanying contractions were obtained to determine the association between electrophysiological and mechanical events. The present study was designed to elucidate the mechanism by which Bay K 8644, caffeine and ryanodine alter force production after variable periods of rest. 3 Bay K 8644 (1 μm) increased steady state contraction in response to a constant train of stimulation, caused rest‐depression after 2 and 8 min rest, prolonged action potential duration and increased action potential plateau amplitude. Augmented steady state tension was not accompanied by any change in time to peak tension or rapid cooling contracture amplitude. However, the post‐rest rapid cooling contracture was greatly diminished compared to that observed prior to Bay K 8644 treatment. 4 Caffeine (3 and 5 mm) caused rest‐depression with an increase in steady state contraction amplitude. Along with this there was a slight decrease in action potential duration and plateau amplitude and an increase in time to peak tension. The rapid cooling contractures were virtually abolished at all conditioning intervals. The effect of caffeine on twitch tension and cooling contracture is consistent with the ability of this compound to inhibit calcium sequestration by the sarcoplasmic reticulum. 5 A combination of Bay K 8644 and caffeine caused significantly less rest‐depression than that seen with Bay K 8644 alone. The augmented twitch tension was accompanied by a long time to peak tension and action potential duration. However, there was no increase in the amplitude of the rapid cooling contracture, either after a regular train of stimulation or after rest, compared to that seen after Bay K 8644. 6 Ryanodine (10 nm), produced rest‐depression, reduced steady state twitch tension and augmented the rest‐depression produced by Bay K 8644. The steady state rapid cooling contracture remained unchanged when both agents were present simultaneously, while the post‐rest rapid cooling contracture was significantly depressed compared to that observed with Bay K 8644 alone. 7 Bay K 8644 and ryanodine appear to have similar actions with respect to promoting diastolic loss of calcium from the sarcoplasmic reticulum. Although caffeine also decreases post‐rest potentiation, it antagonizes rest‐depression caused by Bay K 8644. The data from these experiments suggest that this reversal is a result of depressed intracellular calcium buffering and enhanced myofilament sensitivity produced by caffeine in the presence of increased transmembrane calcium influx promoted by Bay K 8644.

Depression of Canine Ventricular Sarcoplasmic Reticulum by the Calcium Channel Agonist, Bay K 8644

Ca channel agonists are novel compounds which increase cardiac contractile force by increasing slow inward Ca current through the sarcolemma (1,2,3). The presently available compounds are dihydropyridine analogs with structural similarities with agents such as nifedipine, which are Ca channel antagonists. The present study resulted from some chance observation which were made with BAY K 8644, a prototype Ca channel agonist first discovered by Schramm et al (4). This compound was being used by us to evaluate an experimental model designed to test the effects of cardioactive agents on the contribution of transsarcolemmal and transsarcoplasmic reticular Ca movement to inotropy. We observed that BAY K 8644, impaired Ca release from the sarcoplasmic reticulum in addition to its well known effect of increasing transsarcolemmal Ca influx.

μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants

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.

The Action of Calcium Channel Agonists on the Mammalian Ventricular Myocardium

Dihydropyridine Ca channel agonists bear close structural resemblance to several Ca channel antagonists. The most extensively investigated agent in this group is BAY k 8644, first described by Schramm and coworkers. 1−4 Other Ca channel agonists are YC170, 5 202–791, 6 H 160–51. 7 R202–791 6 and CGP28–392. 8,9 The characteristic property of these agents is to increase the ‘open time’ of the ‘L’ type of calcium channels resulting either in an increase in calcium current during an action potential or during depolarisation by other means. 10 The cardiovascular consequence of this is an increase in cardiac contractility and vasoconstriction. 2 It is this latter property which has precluded the clinical use of calcium channel agonists in heart failure. However, the calcium channel agonists hold an important place as pharmacological tools. This review will consider some of the anomalous actions of BAY k 8644 which result in “negative” inotropy under certain circumstances. A possible effect of these drugs on sarcoplasmic reticular function, in addition to their better known effect of enhancing trans-sarcolemmal calcium current, will be discussed.