Anthony H. Caswell

Visualization of membrane bound cations by a fluorescent technique

Abstract Chlorotetracycline binds to divalent diamagnetic cations in aqueous solution with enhanced fluorescence. The fluorescence of the chelate is polarity dependent, being higher in nonaqueous solvents. If biological membranes or detergent micelles which bind cations are suspended in an aqueous phase, the fluorescence of the chlorotetracycline chelate shows enhanced fluorescence over the aqueous complex indicating migration of the divalent cation and chlorotetracycline into the nonaqueous membrane region. The fluorescence of chlorotetracycline bound to mitochondria varies as a function of the respiratory state of the mitochondria.

Kinetics of transport of divalent cations across sarcoplasmic reticulum vesicles induced by ionophores

Abstract The fluorescent chelate probe technique is employed in exploring the kinetics of Ca++ and Sr++ release from sarcoplasmic reticulum induced by two divalent cation ionophores, A23187 and X537A. Both reagents are shown to release Ca++ that has been previously accumulated. A23187 is 60X more effective than X537A, while X537A is 100X more effective than A23187 in sequestering Ca++ into an organic phase in two phase systems. An explanation for the discrepancy between the effectiveness of the antibiotics in the two phase system and the transport rates in sarcoplasmic reticulum is provided by the observation of a particularly low affinity of X537A for Ca++ and Sr++ in polar media. The fluorescence properties of A23187 in the vesicle membrane are described.

Methods of Measuring Intracellular Calcium

Publisher Summary This chapter discusses the methods of measuring intracellular calcium. The techniques described offer a wide range of approaches for assaying intracellular Ca 2+ . The variety of problems involved in monitoring in vivo Ca diffusion among compartments in the short period of the physiological event present peculiar technical challenges Techniques have kept pace with the problems, and at the same time the problems have stimulated development of the techniques. Thus, optical methods for following rapid Ca 2+ transients have been developed over the past few years, and the search continues for more sensitive and selective dyes. The emphasis on the location of Ca 2+ may change from the assay of cytoplasmic Ca2 + toward determination of the source of Ca 2+ which causes alteration of cytoplasmic Ca 2+ and toward the binding site of Ca 2+ which serves to produce the response. In some cells this development has already occurred. The technical problems in monitoring Ca 2+ fluxes within organelles of cells may be considerable, although some progress has already been made.

Selectivity of cation chelation to tetracyclines: evidence for special conformation of calcium chelate.

Abstract Tetracycline antibiotics in apolar solvents chelate to Ca in a different conformation from that of the Mg chelate. Evidence for this different conformation is adduced from the fluorescence, absorption and circular dichroism spectra of the antibiotic bound to Ca and Mg. The conformation of the antibiotic chelated to Ca is a high affinity form. Only those divalent cations of a size similar to or greater than that of Ca are able to induce this conformation. It is tentatively proposed that liganding occurs between both the A ring and the BCD ring conjugated system.

The Migration of Divalent Cations in Mitochondria Visualized by a Fluorescent Chelate Probe

SummaryThe use of the fluorescent chelate probe, chlorotetracycline, in mitochondria is described. The probe shows a high fluorescence in the presence of mitochondria which may be ascribed to binding of the probe to membrane-associated Ca++ and Mg++. The fluorescence excitation and emission spectra are diagnostic of binding of the probe to Ca++ in coupled mitochondria and Mg++ in uncoupled mitochondria. The fluorescence polarization spectra are diagnostic of the cations having a moderately high mobility in the membrane environment. The effects of exogenous EDTA and of endogenous Mn++ indicate that the probe is primarily visualizing actively accumulated Ca++ on the inner surface of the inner membrane. By employing the Ca++ transport inhibitor, Tb+++, the fluorescence changes associated with metabolic alterations are shown to arise partly from cation transport and partly through alterations in the binding properties of the inner surface of the membrane. Chlorotetracycline is a probe for divalent cations associated with the membrane and is of general utility in the study of cation migrations in cellular and subcellular systems.

Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads

SummaryIsolated triadic proteins were employed to investigate the molecular architecture of the triad junction in skeletal muscle. Immunoaffinity-purified junctional foot protein (JFP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase and partially purified dihydropyridine (DHP) receptor were employed to probe protein-protein interactions using affinity chromatography, protein overlay and crosslinking techniques. The JFP, an integral protein of the sarcoplasmic reticulum (SR) preferentially binds to GAPDH and aldolase, peripheral proteins of the transverse (T)-tubule. No direct binding of JFP to the DHP receptor was detected. The interactions of JFP with GAPDH and aldolase appear to be specific since other glycolytic enzymes associated with membranes do not bind to the JFP. The DHP receptor, an integral protein of the T-tubule, also binds GAPDH and aldolase. A ternary complex between the JFP and the DHP receptor can be formed in the presence of GAPDH. In addition, the DHP receptor binds to a previously undetectedMr 95 K protein which is distinct from the SR Ca2+ pump and phosphorylaseb. TheMr 95 K protein is an integral protein of the junctional domain of the SR terminal cisternae. It is also present in the newly identified “strong triads” (accompanying paper). From these findings, we propose a new model for the triad junction.

Ouabain-binding vesicles from skeletal muscle.

Abstract The intact sacrospinalis (back) muscle of a rabbit was injected with [ 3 H]ouabain and allowed to incubate for 20 min. The muscle was excised and homogenized and a microsomal preparation was made. This was placed on a sucrose density gradient and two protein bands were observed at 30 and 40% sucrose regions. Most of the [ 3 H]ouabain was associated with the dense band. If the dense band was loaded with Ca oxalate and placed on a density gradient, then both protein and ouabain migrated equally to the bottom of the gradient. If the 40% band was treated with EDTA, both protein and ouabain migrated to 33% sucrose. It was therefore concluded that ouabain-binding vesicles were associated with the 40% band. If the 40% band was loaded with Ca oxalate and then passed through a French press and placed again on a gradient, the ouabain migrated to 22% sucrose while the protein bands appeared at 17 and 32% sucrose and in the pellet. It was concluded that ouabain-binding vesicles were distinct from the major vesicle population, but were mechanically linked until the linkage was destroyed by mechanical disruption. Electron microscopy revealed that the dense band of the gradient contained terminal cisternae and transverse tubules attached to the terminal cisternae in triad or diad junctions. It was concluded that the 40% band consisted mainly of terminal cisternae and that the ouabain-binding vesicles were transverse tubules attached to terminal cisternae. Transverse tubules therefore may be identified specifically after homogenization.

Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca2+ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca2+ transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca2+.[3H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca2+ activation or Ca2+ and Mg2+ inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca2+ homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.

Observation of calcium uptake by isolated sarcoplasmic reticulum employing a fluorescent chelate probe

Abstract The fluorescent chelate probe technique is employed to observe the accumulation and binding of Ca ++ to isolated sarcoplasmic reticulum from skeletal and cardiac muscle. Chlorotetracycline serves as a fluorescent chelate probe which chelates to membrane bound Ca ++ giving rise to an intensely fluorescence adduct. An increase in fluorescence of chlorotetracycline is caused by ATP induced Ca ++ transport in both skeletal and cardiac muscle microsomes. The fluorescence spectra indicate that Ca ++ lies on the membrane surface in a relatively polar environment.

Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction

SummaryThe Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the Junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m) [125I]iodophenyl diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to Mr, 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa frament was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin