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Calvin C Hale

Publications and source records attributed to Calvin C Hale.

4 recordsLinked to original sources
PubMed2002

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.

Amino Acid Motifs↗
PubMed2002

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.

Animals↗
PubMed2002

The cardiac sodium-calcium exchanger associates with caveolin-3.

The cardiac Na/Ca exchanger's (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 beta-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 transporter's 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.

Amino Acid Sequence↗