Biomedical subjects

K D Philipson

Publications and source records attributed to K D Philipson.

At least 19 recordsLinked to original sources

PubMed2002

Immunohistochemical detection of the sodium-calcium exchanger in rat hippocampus cultures using subtype-specific antibodies.

All of the known Na+/Ca2+ exchanger subtypes, NCX1-3, are expressed in the brain, albeit with marked regional differences. On the mRNA level, overall expression seems most prominent for NCX2, intermediate for NCX1, and, except for a few regions, low for NCX3. Using three subtype-specific antibodies, we have now studied the cellular expression of the NCX subtypes in rat hippocampus cultures by immunohistochemical techniques. Our results provide evidence for a highly cell-specific expression pattern of NCX subtypes and show surprisingly little colocalization. NCX1 and NCX3 are both primarily expressed in neuronal cells. While NCX1 is found in the large majority of neurons, NCX3 expression was restricted to a small minority of cells. By contrast, NCX2 was almost exclusively present in glial cells. The NCX2 antibody, a IgM, stained glial cell membranes as well as an intermediate fibrillar system. In spite of extensive screening, the nature of this fiber system has not yet been identified.

Animals↗

PubMed2001

Gender influences [Ca(2+)](i) during metabolic inhibition in myocytes overexpressing the Na(+)-Ca(2+) exchanger.

BACKGROUND: The Na(+)-Ca(2+) exchanger (NCX) may contribute to Ca(2+) overload and injury in ischemic cardiomyocytes. Recently, NCX overexpression was reported to increase ischemia/reperfusion injury in male and oophorectomized female but not in female mice. We therefore measured the effects of gender and estrogen on [Ca(2+)](i) and [Na(+)](i) during metabolic inhibition (MI) in myocytes from wild-type (WT) and transgenic (TG) mice overexpressing NCX. METHODS AND RESULTS: Flow cytometry was used with fluo 3 for [Ca(2+)](i) and sodium green for [Na(+)](i) measurements. Male TG mouse myocytes had higher [Ca(2+)](i) after 30 minutes of MI (1086+/-160 nmol/L, n=8) than male WT (688+/-104 nmol/L, n=9, P=0.01). The increase in [Ca(2+)](i) during MI induced by NCX overexpression in female myocytes was not significant, however (TG 552+/-62 nmol/L, n=9; WT 426+/-44 nmol/L, n=7). The magnitude of rise in [Ca(2+)](i) during MI was greater in male than female myocytes. KB-R7943, an NCX inhibitor, abolished the effect of NCX overexpression but did not totally eliminate the effect of gender on [Ca(2+)](i) during MI. NCX current density and basal Na(+) pump function were not influenced by gender. The rise in [Na(+)](i) during MI was greater in male than in female myocytes. Estrogen attenuated the increase in [Ca(2+)](i) and [Na(+)](i) in male myocytes during MI and abolished the gender difference in [Na(+)](i) during MI. CONCLUSIONS: Increased expression of NCX results in a more marked rise in [Ca(2+)](i) during MI in male than in female mouse myocytes. This gender difference appears to be mediated in part by an inhibitory effect of estrogen on the rise in [Na(+)](i), an NCX modifier, during MI.

Animals↗

PubMed2001

A disulfide bond is required for functional assembly of NCX1 from complementary fragments.

The cardiac Na(+)-Ca(2+) exchanger consists of a single polypeptide with two transmembrane segment (TMS) clusters separated by a large intracellular loop between TMS5 and TMS6 (Nicoll et al. (1999) J. Biol. Chem. 274, 910-917; Iwamoto et al. (1999) FEBS Lett. 446, 264-268). A "split" exchanger can be expressed by dividing the exchanger cDNA into two fragments so that the NH(2)- and CO(2)H-terminal portions of the protein are expressed as separate polypeptides in HEK293 cells. Expression of partial exchanger molecules did not result in detectable exchanger activity. Cells coexpressing both portions of the exchanger, however, displayed between 30 and 50% of the activity of the intact wild-type exchanger. The full-length exchanger contains a disulfide bond between residues 14 or 20 and 792. We examined the role of this disulfide bond in the split exchanger by mutagenesis and expression studies. Our results indicate that the function of the exchanger requires both TMS clusters and that the C(14 or 20)/C792 disulfide bond is essential for expression of active exchangers from half molecules.

Animals↗

PubMed2001

Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3.

We describe here the identification and characterization of a novel member of the family of K(+)-dependent Na(+)/Ca(2+) exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "alpha -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K(+)-dependent Na(+)/Ca(2+) exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive (45)Ca(2+) uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K(+)-dependent Na(+)/Ca(2+) exchanger family and suggests this class of transporter has a more widespread role in cellular Ca(2+) handling than previously appreciated.

Animals↗

PubMed2001

Split Na+-Ca2+ exchangers. Implications for function and expression.

The Na(+)-Ca(2+) exchanger has nine transmembrane segments, with a large cytoplasmic loop between the fifth and sixth transmembrane segments. The protein was split within the cytoplasmic loop into two domains consisting of the first five transmembrane segments and the last four transmembrane segments, respectively. The two domains were either expressed individually or coexpressed. Each of the two domains with different lengths of the cytoplasmic loop was fused to green fluorescent protein. We show that coexpression of both domains is required for proper membrane targeting and for expression of functional exchange activity. Fusion to green fluorescent protein does not alter biophysical properties of the exchange process. In addition, truncation of a large portion of the cytoplasmic loop does not alter important properties of the exchanger such as Na(+)-dependent inactivation, activation by chymotrypsin, or exchanger inhibitory peptide (XIP) sensitivity.

Animals↗

PubMed2001

Helix packing of functionally important regions of the cardiac Na(+)-Ca(2+) exchanger.

In a revised topological model of the cardiac Na(+)-Ca(2+) exchanger, there are nine transmembrane segments (TMSs) and two possible re-entrant loops (Nicoll, D. A., Ottolia, M., Lu, Y., Lu, L., and Philipson, K. D. (1999) J. Biol. Chem. 274, 910-917; Iwamoto, T., Nakamura, T. Y., Pan, Y., Uehara, A., Imanaga, I., and Shigekawa, M. (1999) FEBS Lett. 446, 264-268). The TMSs form two clusters separated by a large intracellular loop between TMS5 and TMS6. We have combined cysteine mutagenesis and oxidative cross-linking to study proximity relationships of TMSs in the exchanger. Pairs of cysteines were reintroduced into a cysteine-less exchanger, one in a TMS in the NH(2)-terminal cluster (TMSs 1-5) and the other in a TMS in the COOH-terminal cluster (TMSs 6-9). The mutant exchanger proteins were expressed in HEK293 cells, and disulfide bond formation between introduced cysteines was analyzed by gel mobility shifts. Western blots showed that S117C/V804C, A122C/Y892C, A151C/T815C, and A151C/A821C mutant proteins migrated at 120 kDa under reducing conditions and displayed a partial mobility shift to 160 kDa under nonreducing conditions. This shift indicates the formation of a disulfide bond between these paired cysteine residues. Copper phenanthroline and the cross-linker N', N'-o-phenylenedimaleimide enhanced the mobility shift to 160 kDa. Our data suggest that TMS7 is close to TMS3 near the intracellular side of the membrane and is in the vicinity of TMS2 near the extracellular surface. Also, TMS2 must adjoin TMS8. This initial packing model of the exchanger brings two functionally important domains in the exchanger, the alpha 1 and alpha 2 repeats, close to each other.

Animals↗

PubMed2001

Overexpression of the Na(+)/Ca(2+) exchanger and inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase in ventricular myocytes from transgenic mice.

BACKGROUND: Myocytes from failing hearts produce slower and smaller Ca(2+) transients associated with reduction in expression of sarcoplasmic reticulum (SR) Ca(2+) ATPase and an overexpression of Na(+)/Ca(2+) exchanger. Since the physiological role of both these proteins is competing for, and removing, Ca(2+) from the cytoplasm, overexpression of the exchanger may compensate for less effective SR Ca(2+) uptake. This study demonstrates this compensatory effect and provides a quantitative description of the results. METHODS: Ventricular myocytes from transgenic mice overexpressing the Na(+)/Ca(2+) exchanger (TR) and nontransgenic littermates (NON) were used. Cell shortening, cytoplasmic [Ca] (using indo-1 AM) and electrophysiological parameters were monitored. RESULTS: TR myocytes displayed faster Ca(2+) transients and twitches compared with NON myocytes. Superfusion with thapsigargin prolonged the time-course of Ca(2+) transients of TR myocytes until these were equal to the ones measured in NON myocytes. The amount of SR Ca(2+)-ATPase (SERCA) inhibition needed to obtain such transients was calculated as a function of V(max) for the Ca(2+) flux via SERCA and found to be 28%. In TR myocytes V(max) for the Ca(2+) flux via Na(+)/Ca(2+)exchange was 240% of NON myocytes. When Ca(2+) transients in TR myocytes were slowed by thapsigargin to similar values to the ones recorded in NON myocytes, SR Ca(2+) content was also correspondingly reduced. CONCLUSIONS: The results suggest that in pathophysiological conditions where there is a reduction in SERCA function, overexpression of Na(+)/Ca(2+) exchanger can compensate and allow normal Ca(2+) homeostasis to be maintained. In mouse ventricular myocytes a 2.4-fold increase in Na(+)/Ca(2+) exchange activity compensates for a reduction in SERCA function by 28% so maintaining the duration of the Ca(2+) transient.

Animals↗

PubMed2001

Allosteric regulation of Na/Ca exchange current by cytosolic Ca in intact cardiac myocytes.

The cardiac sarcolemmal Na-Ca exchanger (NCX) is allosterically regulated by [Ca](i) such that when [Ca](i) is low, NCX current (I(NCX)) deactivates. In this study, we used membrane potential (E(m)) and I(NCX) to control Ca entry into and Ca efflux from intact cardiac myocytes to investigate whether this allosteric regulation (Ca activation) occurs with [Ca](i) in the physiological range. In the absence of Ca activation, the electrochemical effect of increasing [Ca](i) would be to increase inward I(NCX) (Ca efflux) and to decrease outward I(NCX). On the other hand, Ca activation would increase I(NCX) in both directions. Thus, we attributed [Ca](i)-dependent increases in outward I(NCX) to allosteric regulation. Ca activation of I(NCX) was observed in ferret myocytes but not in wild-type mouse myocytes, suggesting that Ca regulation of NCX may be species dependent. We also studied transgenic mouse myocytes overexpressing either normal canine NCX or this same canine NCX lacking Ca regulation (Delta680-685). Animals with the normal canine NCX transgene showed Ca activation, whereas animals with the mutant transgene did not, confirming the role of this region in the process. In native ferret cells and in mice with expressed canine NCX, allosteric regulation by Ca occurs under physiological conditions (K(mCaAct) = 125 +/- 16 nM SEM approximately resting [Ca](i)). This, along with the observation that no delay was observed between measured [Ca](i) and activation of I(NCX) under our conditions, suggests that beat to beat changes in NCX function can occur in vivo. These changes in the I(NCX) activation state may influence SR Ca load and resting [Ca](i), helping to fine tune Ca influx and efflux from cells under both normal and pathophysiological conditions. Our failure to observe Ca activation in mouse myocytes may be due to either the extent of Ca regulation or to a difference in K(mCaAct) from other species. Model predictions for Ca activation, on which our estimates of K(mCaAct) are based, confirm that Ca activation strongly influences outward I(NCX), explaining why it increases rather than declines with increasing [Ca](i).

Allosteric Regulation↗

PubMed2001

Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor.

The nonsense codon suppression technique was used to incorporate o-nitrobenzyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9' position of the M2 transmembrane segment of the gamma-subunit of the muscle nicotinic ACh receptor expressed in Xenopus oocytes. The caged amino acids replaced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradiation to remove the caging group. This represents the first successful incorporation of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. The bulky nitrobenzyl group does not prevent the synthesis, assembly, or trafficking of the ACh receptor. When side chains were decaged using 1-ms UV light flashes, the channels with caged Cys or caged Tyr responded with strikingly different kinetics. The increase in current upon photolysis of caged Cys was too rapid for resolution by the voltage-clamp circuit [time constant (tau) <10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with tau approximately 500 ms. Apparently, the presence of a bulky o-nitrobenzyl Tyr residue distorts the receptor into an abnormal conformation. Upon release of the caging group, the receptor relaxes, with tau approximately 500 ms, into a conformation that allows the channel to open. Tyr at the 9' position of the gamma-subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. This is manifested in two ways. 1) A "rebound," or increase in current, occurs upon removal of ACh from the bathing medium; and 2) at ACh concentrations >400 microM, inward currents are decreased through the mutated channel. The ability to incorporate caged amino acids into proteins should have widespread utility.

Animals↗

PubMed2000

Impaired contractile performance of cultured rabbit ventricular myocytes after adenoviral gene transfer of Na(+)-Ca(2+) exchanger.

Na(+)-Ca(2+) exchanger (NCX) gene expression is increased in the failing human heart. We investigated the hypothesis that upregulation of NCX can induce depressed contractile performance. Overexpression of NCX was achieved in isolated rabbit ventricular myocytes through adenoviral gene transfer (Ad-NCX). After 48 hours, immunoblots revealed a virus dose-dependent increase in NCX protein. Adenoviral beta-galactosidase transfection served as a control. The fractional shortening (FS) of electrically stimulated myocytes was analyzed. At 60 min(-1), FS was depressed by 15.6% in the Ad-NCX group (n=143) versus the control group (n=163, P:<0.05). Analysis of the shortening-frequency relationship showed a steady increase in FS in the control myocytes (n=26) from 0.027+/-0.002 at 30 min(-1) to 0. 037+/-0.002 at 120 min(-1) (P:<0.05 versus 30 min(-1)) and to 0. 040+/-0.002 at 180 min(-1) (P:<0.05 versus 30 min(-1)). Frequency potentiation of shortening was blunted in NCX-transfected myocytes (n=27). The FS was 0.024+/-0.002 at 30 min(-1), 0.029+/-0.002 at 120 min(-1) (P:<0.05 versus 30 min(-1), P:<0.05 versus control), and 0. 026+/-0.002 at 180 min(-1) (NS versus 30 min(-1), P:<0.05 versus control). Caffeine contractures, which indicate sarcoplasmic reticulum Ca(2+) load, were significantly reduced at 120 min(-1) in NCX-transfected cells. An analysis of postrest behavior showed a decay of FS with longer rest intervals in control cells. Rest decay was significantly higher in the Ad-NCX group; after 120 seconds of rest, FS was 78+/-4% in control and 65+/-3% in the Ad-NCX group (P:<0.05) relative to steady-state FS before rest (100%). In conclusion, the overexpression of NCX in rabbit cardiomyocytes results in the depression of contractile function. This supports the hypothesis that upregulation of NCX can result in systolic myocardial failure.

Adenoviridae↗

PubMed2000

Functional analysis of a disulfide bond in the cardiac Na(+)-Ca(2+) exchanger.

The electrophoretic mobility of the cardiac Na(+)-Ca(2+) exchange protein is different under reducing and nonreducing conditions. This mobility shift is eliminated in a cysteine-less exchanger, suggesting that the presence or absence of an intramolecular disulfide bond alters the conformation and mobility of the exchanger. Using cysteine mutagenesis and biochemical analysis, we have identified the cysteine residues involved in the disulfide bond. Cysteine 792 in loop h of the exchanger forms a disulfide bond with either cysteine 14 or 20 near the NH(2) terminus. Because the NH(2) terminus is extracellular, the data establish that loop h must also be extracellular. A rearrangement of disulfide bonds has previously been implicated in the stimulation of exchange activity by combinations of reducing and oxidizing agents. We have investigated the role of cysteines in the stimulation of the exchanger by the combination of FeSO(4) and dithiothreitol (Fe-DTT). Using the giant excised patch technique, we find that stimulation of the wild type exchanger by Fe-DTT is primarily due to the removal of a Na(+)-dependent inactivation process. Analysis of mutated exchangers, however, indicates that cysteines are not responsible for stimulation of the exchange activity by Fe-DTT. Ca(2+) blocks modification of the exchanger by Fe-DTT. Disulfide bonds are not involved in redox stimulation of the exchanger, and the modification reaction is unknown. Modulation of Na(+)-dependent inactivation may be a general mechanism for regulation of Na(+)-Ca(2+) exchange activity and may have physiological significance.

Cysteine↗

PubMed2000

Sodium-calcium exchange: a molecular perspective.

Plasma membrane Na(+)-Ca2+ exchange is an essential component of Ca2+ signaling pathways in several tissues. Activity is especially high in the heart where the exchanger is an important regulator of contractility. An expanding exchanger superfamily includes three mammalian Na(+)-Ca2+ exchanger genes and a number of alternative splicing products. New information indicates that the exchanger protein has nine transmembrane segments. The exchanger, which transports Na+ and Ca2+, is also regulated by these substrates. Some molecular information is available on regulation by Na+ and Ca2+ and by PIP2 and phosphorylation. Altered expression of the exchanger in pathophysiological states may contribute to various cardiac phenotypes. Use of transgenic approaches is beginning to improve our knowledge of exchanger function.

Amino Acid Sequence↗

PubMed2000

Interaction of PIP(2) with the XIP region of the cardiac Na/Ca exchanger.

The sarcolemmal Na/Ca exchanger undergoes an inactivation process in which exchange activity decays over several seconds following activation by the application of Na to the intracellular surface of the protein. Inactivation is eliminated by an increase in membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). Inactivation is also strongly affected by mutations to a basic 20-amino acid segment of the exchanger known as the endogenous XIP region. The hypothesis that PIP(2) directly interacts with the XIP region of the exchanger was tested. First, we investigated the ability of a peptide with the same sequence as the XIP region to bind to immobilized phospholipid vesicles. (125)I-labeled XIP bound avidly to vesicles containing only a low concentration (<3%) of PIP(2). The binding was specific, in that binding was not displaced by other basic peptides. The effects of altering the sequence of XIP peptides also indicated binding specificity. Second, we examined the functional response to PIP(2) of exchangers with mutated XIP regions. Outward Na/Ca exchange currents were measured using the giant excised patch technique. The mutated exchangers either had no inactivation or accelerated inactivation. In both cases, the exchangers no longer responded to PIP(2) or to PIP(2) antibodies. Overall, the data indicate that the affinity of the endogenous XIP region for PIP(2) is an important determinant of the inactivation process.

Animals↗

PubMed2000

Enhanced gene expression of Na(+)/Ca(2+) exchanger attenuates ischemic and hypoxic contractile dysfunction.

Enhanced gene expression of the Na(+)/Ca(2+) exchanger in failing hearts may be a compensatory mechanism to promote influx and efflux of Ca(2+), despite impairment of the sarcoplasmic reticulum (SR). To explore this, we monitored intracellular calcium (Ca(i)(2+)) and cardiac function in mouse hearts engineered to overexpress the Na(+)/Ca(2+) exchanger and subjected to ischemia and hypoxia, conditions known to impair SR Ca(i)(2+) transport and contractility. Although baseline Ca(i)(2+) and function were similar between transgenic and wild-type hearts, significant differences were observed during ischemia and hypoxia. During early ischemia, Ca(i)(2+) was preserved in transgenic hearts but significantly altered in wild-type hearts. Transgenic hearts maintained 40% of pressure-generating capacity during early ischemia, whereas wild-type hearts maintained only 25% (P < 0.01). During hypoxia, neither peak nor diastolic Ca(i)(2+) decreased in transgenic hearts. In contrast, both peak and diastolic Ca(i)(2+) decreased significantly in wild-type hearts. The decline of Ca(i)(2+) was abbreviated in hypoxic transgenic hearts but prolonged in wild-type hearts. Peak systolic pressure decreased by nearly 10% in hypoxic transgenic hearts and >25% in wild-type hearts (P < 0.001). These data demonstrate that enhanced gene expression of the Na(+)/Ca(2+) exchanger preserves Ca(i)(2+) homeostasis during ischemia and hypoxia, thereby preserving cardiac function in the acutely failing heart.

Animals↗

PubMed2000

Functional properties of transgenic mouse hearts overexpressing both calsequestrin and the Na(+)-Ca(2+) exchanger.

Overexpression of calsequestrin (CSQ) induces severe cardiac hypertrophy, whereas overexpression of Na(+)-Ca(2+) exchanger (NCX) does not affect cardiac weight. To investigate a possible beneficial effect of NCX in hypertrophy, we produced transgenic mice overexpressing both NCX and CSQ (NCX/CSQ). Surprisingly, these mice developed severe heart failure. The heart/body weight ratio was enhanced and the mRNA expression of ANF, as a marker of hypertrophy, was highest in double transgenic mice. In isolated muscle strips, the basal relaxation time was prolonged in CSQ and NCX/CSQ mice. Moreover, in the presence of caffeine, force of contraction was increased only in CSQ and NCX/CSQ and was accompanied by elevated diastolic tension. In some respects, however, additional overexpression of NCX altered the CSQ phenotype into the wild-type phenotype. The expression of sarcoplasmic reticulum (SR)-Ca(2+)-ATPase and phospholamban, proteins involved in the Ca(2+) uptake of the SR, were only increased in CSQ, indicating a possible influence of NCX in the regulation of SR-Ca(2+) uptake proteins. The Ca(2+) transients and the L-type Ca(2+) currents in the presence of caffeine were very large in CSQ, but smaller increases were noted in double transgenic mice. Therefore, the successful co-overexpression of CSQ and NCX in these mice provides a novel model in which to investigate the interaction of proteins tightly linked to maintain Ca(2+) homeostasis.

Animals↗

PubMed1999

A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger.

The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+ gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.

Animals↗

PubMed1999

Quantitation of Na/Ca exchanger function in single ventricular myocytes.

A Na/Ca exchange current can be elicited in voltage clamped single ventricular myocytes by the abrupt removal of extracellular Na+ by means of a rapid switcher device. We measured this reverse Na/Ca exchange current in isolated mouse ventricular myocytes from wild-type mice, and from transgenic mice with hearts overexpressing the Na/Ca exchanger. In mouse ventricular myocytes, the current was sensitive to nickel, and was eliminated by removal of intracellular Na+. It was not influenced by 3 m m ouabain, and thus not contaminated by Na pump currents. The magnitude of the current reached a plateau within 10-15 min after obtaining a whole cell patch with the pipettes containing EGTA, to buffer [Ca2+]i and in zero extracellular K+ concentration to completely inhibit the Na pump, and allow equilibration of pipette Na+ with subsarcolemmal [Na+]. The magnitude of the current increased with increases in pipette [Na+]. Comparison of the current magnitudes in wild-type and transgenic myocytes showed a 2.5 and 2.7 fold increase in the current in transgenic myocytes at pipette [Na+] of 10 and 20 m m. The magnitude of this increase in Na/Ca exchanger currents in single transgenic myocytes compares well with the reported 2.5 fold increase in Na+-dependent 45Ca2+ uptake measured in ventricular sarcolemmal vesicles obtained from transgenic animals. With this approach, we found variation in exchanger current densities in different species, with values for mouse>rat>rabbit>dog>human. This technique should also be useful in quantifying changes in Na/Ca exchanger current density as a consequence of pathologic processes, and exposure to drugs.

Animals↗

PubMed1999

Energetics of Na(+)-Ca(2+) exchange in resting cardiac muscle.

The energetic effect of extracellular Na(+) removal and readmission (in a nominally Ca(2+)-free perfusate) in Langendorff-perfused ventricles of transgenic mice (TM), which overexpress the sarcolemmal Na(+)-Ca(2+) exchanger; normal mice (NM); young (7-12 days old) rats (YR); and older (13-20 days old) rats (OR) was studied. In all heart muscles, extracellular Na(+) removal induced an increase in heat production (H(1)). Na(+) readmission further increased heat production to a peak value (H(2)) followed by a decrease toward initial values. These effects were more marked in the YR and TM as compared with the OR and NM groups, respectively. Caffeine (1 mM), ryanodine (0.2 microM), and verapamil (1 microM) decreased H(1) and H(2) in both rat groups. EGTA (1 mM) decreased H(1) and H(2) in the YR but not in the OR group. Thapsigargin (1 microM) decreased H(1) and H(2) in all four hearts preparations. A possible interpretation is that Na(+)-Ca(2+) exchange acts as an energy-saving mechanism to prevent Ca(2+) accumulation at the junctional sarcoplasmic reticulum zone (JSR) and thus prevents further release of Ca(2+). Extracellular Na(+) removal lead to Ca(2+) accumulation in the JSR inducing further SR-Ca(2+) release and increased energy release. Na(+) readmission removes the accumulated Ca(2+) at the JSR (cleft) zone by exchanging Ca(2+) with Na(+) producing a transitory increase in energy release due to Na(+)-K pump activation.

Aging↗