Exchanger Inhibitory Peptide Research Articles

Studies of the mechanism underlying increased Na +/Ca 2+ exchange activity in Alzheimer's disease brain

The Na +/Ca 2+ exchanger was characterized in plasma membrane vesicles derived from frozen hustmortem tissues. The frontal cortex, temporal cortex and cerebellum of control and Alzheimer's disease (AD) tissues were compared. Na +/Ca 2+ exchange activity was defined as the change in vesicular Ca 2+ content seen after Na + loaded vesicles were diluted into choline buffer. The time course of changes in Ca 2+ content after dilution was similar in all three regions of control brain. In AD brain, both frontal and temporal cortex vesicles showed elevated Ca 2+ content, most evident as an increased peak Ca 2+ content at 2 min. The AD cerebellar cortex time course was similar to control and did not show an elevated peak at 2 min. No differences were seen in the passive permeability to Ca 2+ when comparing plasma membrane vesicles prepared from control and AD brain. Vesicles from the frontal and temporal cortex of AD brain showed increases in the V max of the initial velocity of Ca 2+ uptake when compared to control brain, whereas, the cerebellum did not. There were no significant effects of AD on the K m for Ca 2+ activation of the initial velocity. Ca 2+ influx measured during the rise in vesicular Ca 2+ content was elevated in vesicles from AD temporal cortex when compared to control. Two known inhibitors (exchange inhibitory peptide and dichlorobenzamil) of the cardiac Na +/Ca 2+ exchanger inhibited the human brain exchanger equally well in control and AD vesicles. Increased Na +/Ca 2+ exchange activity was not due to astrocytic gliosis. An antibody to the cardiac exchanger was used to determine the molecular weight of the human brain Na +/Ca 2+ exchanger. The molecular weight determinations from Western blots showed identical molecular weights of 100–110 kDa in both AD and control vesicle preparations. These data are entirely consistent with the proposal that increased Na +/Ca 2+ exchange activity in AD reflects the properties of surviving neurons with elevated Na +/Ca 2+ × exchange capacity and lend support to the ‘Ca 2+ hypothesis of AD’.

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

The inhibitory effects of Na+/Ca2+ exchange inhibitory peptide (XIP), which corresponds to residues 219-238 of the Na+/Ca2+ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na(+)-dependent Ca2+ uptake in both rat brain [IC50 = 3.05 +/- 0.69 microM (mean +/- SE)] and human brain (IC50 = 3.58 +/- 0.58 microM). The maximal inhibition seen in rat brain vesicles was approximately 80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na(+)-dependent Ca2+ release, and the inhibitory effect was enhanced by increasing the extravesicular Na+ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na+. When XIP was added at steady state (5 min after the initiation of intravesicular Na(+)-dependent Ca2+ uptake), it was found that the intravesicular Ca2+ content declined with time. Analysis of unidirectional fluxes for Ca2+ at steady state showed that 50 microM XIP inhibited Ca2+ influx and efflux approximately 85 and 70%, respectively. This result suggested that XIP inhibited both Na+/Ca2+ exchange and Ca2+/Ca2+ exchange but had no effect on the passive release pathway for Ca2+. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na+ or other monovalent cations, e.g., K+, is required for XIP to have its inhibitory effect on Ca2+ transport.

Cardiac sarcolemmal Na/Ca-inhibiting peptides XIP and FMRF-amide also inhibit Na/Ca exchange in squid axons

The effect of two cardiac sarcolemmal inhibitory peptides, the 20-amino acid exchange inhibitory peptide (XIP) and the molluscan cardioexcitatory tetrapeptide amide Phe-Met-Arg-Phe-NH2 (FMRFa), were tested in dialyzed squid giant axons. XIP injected into axons causes a maximal inhibition of 52 +/- 8% (n = 6) in the external Na (Nao)-dependent Ca efflux. The inhibitory effect was the same in axons dialyzed with saturating intracellular Ca (Cai) concentration (100 microM) and no MgATP or in axons containing submicromolar Cai concentrations (0.7 microM) and 2 mM MgATP. FMRFa, a peptide that shows no obvious homology with XIP, also causes a marked inhibition in Nao-dependent Ca efflux. As in cardiac sarcolemmal vesicles, the peptide inhibits with low apparent affinity (Ki = 1.9 microM; n = 5). Like XIP, FMRFa has the same effect in axons dialyzed with or without MgATP. The data indicate that XIP, which resembles an endogenous calmodulin binding site that may have an autoregulatory function, and the tetrapeptide FM-RFa, which binds to a putative opiate site, both inhibit Na/Ca exchange in squid axons. The sites at which these peptides bind are not related to the nucleotide (MgATP) regulation of Na/Ca exchange. We therefore suggest that these two sites in the vertebrate cardiac Na/Ca exchange are conserved in the invertebrate axon exchanger.

Interaction of cardiac Na-Ca exchanger and exchange inhibitory peptide with membrane phospholipids.

We tested the hypothesis that the exchange inhibitory peptide (XIP) domain in the cardiac Na-Ca exchanger is a regulatory site under the control of the membrane lipid environment. We found that 125I-XIP bound to liposomes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) with peak binding at 1:1 PC/PS. No binding was observed in PC liposomes. XIP and pentalysine-inhibitable bovine sarcolemmal (SL) Na-Ca exchange activity was observed in reconstituted proteoliposomes composed of 1:1 PC/PS. Proteolysis of SL membranes resulted in a twofold stimulation of Na-Ca exchange activity, but the half-maximal inhibitory concentration (IC50) for XIP (3 microM) was not significantly changed, suggesting that the XIP binding site remained intact. In contrast, the IC50 for pentalysine was decreased from 500 to 150 microM in proteolyzed membranes. These data are consistent with a model of Na-Ca exchange regulation in which the endogenous XIP domain interacts either with another region of the exchange protein to induce an inactive conformational state or with membrane lipid to produce an active conformation.

Relation between reverse sodium-calcium exchange and sarcoplasmic reticulum calcium release in guinea pig ventricular cells.

Exchange-inhibitory peptide (XIP) can inhibit sodium-calcium exchange without inhibiting L-type calcium current (ICa). We therefore used this compound to test the hypothesis that reverse sodium-calcium exchange can trigger contraction in guinea pig ventricular myocytes. When cells were dialyzed with 20 mmol/L sodium, rapid blockade of ICa with nifedipine had little effect on cell shortening. However, if reverse exchange was inhibited by first dialyzing the cells with XIP, blockade of ICa largely inhibited cell shortening. In cells dialyzed with 10 mmol/L sodium, about 51% of the maximum cell shortening remained after ICa was blocked. When both ICa and reverse exchange were significantly inhibited with nifedipine and XIP, only 24% of the cell shortening remained; ie, 27% was XIP inhibitable. Cells dialyzed with solutions deficient in sodium exhibited contractions that were largely dependent on ICa (ie, not XIP inhibitable). If the sarcoplasmic reticulum (SR) was disabled with ryanodine and thapsigargin, reverse exchange could not cause contraction. We therefore conclude that with intact SR, reverse sodium-calcium exchange activates contraction by triggering calcium release from the SR in cells dialyzed with either 10 or 20 mmol/L sodium. A scrambled sequence of XIP, sXIP, caused no measurable effect on contraction.

Initial localization of regulatory regions of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

We have analyzed the regulatory properties of the wild-type cardiac Na(+)-Ca2+ exchanger expressed in Xenopus laevis oocytes using the giant excised patch technique. The exchanger is activated by cytoplasmic application of chymotrypsin and exhibits a number of properties that can be changed or abolished by chymotrypsin treatment, including cytoplasmic Na(+)-dependent inactivation, secondary regulation by free cytoplasmic Ca2+, and inhibition by exchanger inhibitory peptide. Thus, the cloned exchanger expressed in oocytes exhibits regulatory properties similar to those of the native sarcolemmal exchanger. The exchanger protein contains a large (520 amino acids) hydrophilic domain modeled to be intracellular. The role of this region in exchanger function and regulation was examined by deletion mutagenesis. Mutants with residues 240-679 and 562-685 deleted exhibited exchange activity, indicating that this extensive region is not essential for transport function. Both mutants were stimulated by chymotrypsin treatment. Neither mutant demonstrated regulation by free cytoplasmic Ca2+ (Ca2+i) or inhibition by exchanger inhibitory peptide (XIP). However, mutant delta 562-685 but not delta 240-679 displayed Na(+)-dependent inactivation. The data suggest that the binding sites for XIP and regulatory Ca2+ may reside in the region encompassed by residues 562-685. A chimera made from renal and cardiac exchangers has normal regulatory characteristics and helps to further define these sites.

Interactions of the exchange inhibitory peptide with Na-Ca exchange in bovine cardiac sarcolemmal vesicles and ferret red cells.

The Na-Ca exchange inhibitory peptide (XIP), which corresponds to residues 251-270 of the Na-Ca exchange protein, specifically inhibits exchange activity (Li, Z., Nicoll, D. A, Collins, A., Hilgemann, D. W., Filoteo, A. G., Penniston, J. T., Weiss, J. N., Tomich, J. M., and Philipson, K. D. (1991) J. Biol. Chem. 266, 1014-1020). We have found that XIP decreased Na+i-dependent Ca2+ uptake to 46 and 20% of control in mixed and inside-out bovine sarcolemmal (SL) vesicles, respectively, and to 22% of control in ferret red cell vesicles. XIP inhibited uptake in bovine SL vesicles after proteolytic digestion. XIP also inhibited Na+o-dependent Ca2+ efflux in bovine SL vesicles but did not inhibit Ca2+ uptake in reconstituted proteoliposomes. Extracellular XIP did not inhibit Ca2+ uptake into intact ferret red cells. Inhibition of uptake in bovine SL vesicles was reduced as the ionic strength was increased. 125I-labeled XIP (1 microM) was cross-linked to proteins of bovine SL vesicles, ferret red cell vesicles, and intact ferret red cells. Labeling of bands at approximately 75, 120, and 220 kDa (in bovine SL vesicles) and bands at 55 and 85 kDa (in ferret red cell vesicles) was detected. No cross-linking was detected in intact ferret red cells. We conclude that XIP inhibition is insensitive to proteolytic digestion and is partially dependent on charge association and conformation of the exchanger. XIP binds to and interacts with the intracellular side of the Na-Ca exchanger.

Identification of a peptide inhibitor of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

The deduced amino acid sequence of the cardiac sarcolemmal Na(+)-Ca2+ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na(+)-Ca2+ exchange activity. The peptide inhibits the Na(+)-dependent Ca2+ uptake (KI approximately 1.5 microM) and the Nao(+)-dependent Ca2+ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na+ and Ca2+. The peptide is also a potent inhibitor (KI approximately 0.1 microM) of the Na(+)-Ca2+ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na(+)-Ca2+ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.