Calvin C. Hale

Evidence for cardiac sodium-calcium exchanger association with caveolin-3.

The interaction of cardiac Na+–Ca2+ exchange (NCX1) with caveolin proteins was investigated in sarcolemmal vesicles. Western blots of sarcolemmal vesicles revealed the presence of caveolin‐1, ‐2, and ‐3. NCX1 co‐fractionated more closely with caveolin‐3 than caveolin‐1 on sucrose density gradients. NCX1 has five possible caveolin‐binding motifs and NCX1 co‐precipitated specifically with caveolin‐3. Molecular sieve column chromatography indicated that this co‐precipitation was not due to incomplete solubilization of lipid raft microdomains. Cholesterol chelation in vesicles decreased NCX1 transport activity and caveolin‐3 co‐precipitation. NCX1 may play a role in caveolar transmembrane signaling in addition to its role in excitation–contraction coupling.

The Cardiac Sodium‐Calcium Exchanger Associates with Caveolin‐3

Abstract: The cardiac Na/Ca exchangers (NCX1) role in calcium homeostasis during myocardial contractility makes it a possible target of signaling factors regulating inotropy. Caveolae, structured invaginations of the plasmalemma, are known to concentrate a wide variety of signaling factors. The predominant coat proteins of caveolae, caveolins, dock to and regulate the activity of these signaling factors and other proteins through interaction with their scaffolding domain. In this study we investigated the interaction of NCX1 with caveolin proteins. Western blots of bovine cardiac sarcolemmal vesicles revealed the presence of caveolin‐1, ‐2, and ‐3. Immunoprecipitation of detergent‐solubilized vesicle proteins with either NCX1 or caveolin‐3 antibodies indicated that NCX1 coprecipitates with caveolin‐3, but not with caveolin‐1 and ‐2. Functional disruption of caveolae, by β‐cyclodextrin treatment of vesicles, diminished coprecipitation of caveolin‐3 and NCX1 activity. NCX1 has five potential caveolin‐binding motifs, two of which are in the transporters exchange inhibitory peptide (XIP) domain. The presence of 50 mM XIP peptide enhanced coprecipitation of caveolin‐3 with NCX1 independent of calcium concentration. We conclude that NCX1 associates specifically with caveolin‐3. Partitioning of NCX1 in caveolae has implications for temporal and spatial regulation of excitation‐contraction and ‐relaxation coupling in cardiac myocytes.

Plasmalogen and anionic phospholipid dependence of the cardiac sarcolemmal sodium-calcium exchanger

Although plasmalogens are the predominant phospholipids of cardiac sarcolemma, their physiological role has not been forthcoming. Since the cardiac sarcolemmal sodium‐calcium exchanger has been proposed to be regulated by anionic phospholipids, the roles of plasmalogens and anionic phospholipids as regulators of the sodium‐calcium exchanger were explored. Reconstituted sodium‐calcium exchange activity in plasmalogen‐containing proteoliposomes was 10‐fold higher than that in control proteoliposomes comprised of only diacyl phospholipids. Additionally, exchange activity in plasmalogen‐containing proteoliposomes was regulated by anionic phospholipids. Thus, plasmalogens provide a critical lipid environment in which anionic phospholipids serve as boundary lipids for the regulation of the trans‐sarcolemmal sodium‐calcium exchanger.

The selective activation of the cardiac sarcolemmal sodium‐calcium exchanger by plasmalogenic phosphatidic acid produced by phospholipase D

Since plasmalogens are the predominant phospholipid of cardiac sarcolemma, the activation of the sodium‐calcium exchanger by either plasmenylethanolamine or plasmalogenic phosphatidic acid generated by phospholipase D was explored. Sodium‐calcium exchange activity was 7‐fold greater in proteoliposomes comprised of plasmenylethanolamine compared to proteoliposomes comprised of only plasmenylcholine. Phospholipase D treatment of proteoliposomes resulted in 1 mol % conversion of plasmenylcholine or phosphatidylcholine to their respective phosphatidic acid molecular species with a concomitant 8‐fold or 2‐fold activation of sodium‐calcium exchange activity, respectfully. Thus, phospholipase D‐mediated hydrolysis of plasmalogens to phosphatidic acid may be an important mechanism for the regulation of the sodium‐calcium exchanger.

Evidence for high molecular weight Na−Ca exchange in cardiac sarcolemmal vesicles

SummaryCardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na−Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na−Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (Mr) of approximately 226,000±20,000sd (n=6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 1∶1 with mobile phase buffer containing 50mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na−Ca exchange activity. Na−Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na−Ca exchange activity were 91±7 and 68±11%, respectively. In the peak fraction, Na−Ca exchange specific activity increased two-to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimumMr of 224,000 Da. Specific45Ca2+-binding SL proteins withMr of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose.These data suggest that native cardiac Na−Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na−Ca exchange protein(s) remains incomplete.

Identification of a 65 kDa endothelin receptor in bovine cardiac sarcolemmal vesicles

Endothelin-1, an endothelial cell-derived vasoconstrictor peptide, also exerts a potent positive inotropic effect on cardiac tissue. Characterization of specific binding of endothelin-1 to bovine cardiac sarcolemmal vesicles is reported. In the presence of 1 mM CaCl2, the observed binding for 125I-endothelin-1 had a Kd of 6.2 nM with an observed Bmax of 14 pmol/mg sarcolemmal protein. In the presence of 1 mM EDTA (and no added Ca2+) Bmax was reduced to 9 pmol/mg sarcolemmal protein while the Kd remained unchanged. Binding affinity for sarafotoxin S6b was at least one order of magnitude less than for endothelin-1. 125I-Endothelin-1 covalently cross-linked to a sarcolemmal protein with an apparent molecular weight of 65 kDa. Site-directed polyclonal antibodies to a sequence located on the third extramembranal segment of a previously cloned endothelin ETA receptor from bovine lung were produced. Using Western blot analysis, the site-directed polyclonal antibody recognized a sarcolemmal protein at 65 kDa. We conclude that sarcolemmal membranes from bovine ventricular myocardium contain an endothelin binding site and that it is a protein with an apparent molecular weight of 65 kDa.

The effect of Li+ on NaCa exchange in cardiac sarcolemmal vesicles

The effects of Li+ on Na-Ca exchange in bovine cardiac sarcolemmal vesicles were examined. The initial rate of Na(+)-dependent Ca2+ uptake and efflux was inhibited by Li+ in a dose dependent manner. The initial rate of Na(+)-dependent Ca2+ uptake was inhibited 49.8 +/- 2.9% (S.E.) (n = 6) in the presence of Li+ compared to activity in external K+ or choline+. Kinetic analysis indicated that Li+ increased the Km for Ca2+ (96.3 microM) compared to K+ and choline+ (25.5 and 22.9 microM respectively) while Vmax (1.4, 1.2 and 1.1 nmol Ca2+/mg protein/sec respectively) remained unchanged. Li+ did not alter the experimentally derived stoichiometry of the exchange reaction of 3 Na+ for 1 Ca2+.

Purification of a basic fibroblast growth factor-binding proteoglycan from bovine cardiac plasma membrane

A heparan sulfate proteoglycan (HSPG) from bovine cardiac plasma membrane was purified to homogeneity using either isoelectric focusing or anion-exchange chromatography, followed by affinity chromatography on immobilized basic fibroblast growth factor (bFGF). Fractions were assayed for bFGF-binding activity using 125I-bFGF as a probe. Purified proteoglycan ran as a broad band on SDS-PAGE, spanning an apparent molecular mass range of 100-200 kDa, and could be incorporated into liposomes. Digestion of radioiodinated proteoglycan with heparitinase yielded a product of 73 kDa, while digestion with chondroitinase ABC did not change the apparent molecular mass. Monoclonal antibody directed against the ectodomain of another plasma membrane HSPG, syndecan, failed to recognize the purified cardiac proteoglycan on immunoblots. We conclude that adult bovine myocardium contains a membrane-associated bFGF-binding heparan sulfate proteoglycan containing little or no chondroitin sulfate and that this HSPG may be distinct from those of the syndecan family of heparan sulfate proteoglycans.

Ion specificity and stoichiometry of the cardiac inositol transporter

Cardiac myocytes have a high affinity. Mg(2+)-dependent, electrogenic, Na-inositol co-transporter on the plasma or sarcolemma (SL) membrane (Rubin and Hale, 1993). The ion specificity and stoichiometry of this transport process is unknown. Using bovine cardiac SL vesicles, we have determined that transmembrane movement of myo-[3H]inositol requires Na+ and is not supported by K+ or Li+. Furthermore, replacing Cl- with a non-permeant anion has no effect on inositol transport. Carrier-mediated inositol efflux indicated that efflux was Na(+)-dependent and electrogenic but independent of extravesicular Mg2+ in the efflux media. The transport stoichiometry of 1 Na+ for one inositol was derived by determining the null point of myo-[3H]inositol flux under conditions where the inside and outside concentration (ratio) of myoinositol and Na+ were controlled. The results suggest that Na+, inositol, and Mg2+ bind to the same side of the membrane for transport to occur and that the stoichiometry of Na+ and inositol transport is 1:1.

Expressing and purifying membrane transport proteins in high yield.

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.