Frederic Mandel

Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding

This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of125I-labeled CaM at both micromolar and nanomolar Ca2+concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.

On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase

Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na+,K+-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na+,K+-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na+,K+-ATPase were used. Dose–response curves for ouabain, a specific Na+,K+-ATPase inhibitor, were obtained to ascertain which Na+,K+-ATPase isoform(s) is involved. The ACh dose–response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na+,K+-ATPase was studied in purified lamb kidney Na+,K+-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably α2) isoform of Na+,K+-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na+,K+-ATPase. Possible interaction mechanisms are discussed.

Macromolecular dimensions obtained by an efficient Monte Carlo method: The mean square end‐to‐end separation

The ’’slithering snake’’ Monte Carlo technique has been used to generate a very large number of samples on the three choice square planar lattice for chains of 20, 40, 60, 100, 180, 360, and 600 links. The corresponding mean square end‐to‐end separations, 〈r2〉, have been calculated with greater precision than previously possible and compared with the earlier Monte Carlo calculations and with the asymptotic formula of Domb. The present Monte Carlo technique has also been used to generate end‐to‐end distributions for long chains. The correlation between samples for the ’’slithering snake’’ method is also discussed.

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756–765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639–641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase α2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase α2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase α subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.

Self‐avoiding walks subject to boundary constraints

A statistical study is carried out for self‐avoiding walks on infintely long square lattice strips, two and three lines wide. The two‐layer problem is solved completely and the three‐layer problem asymptotically. In each instance, the mean square end‐to‐end separation of a walk is found, as expected, to be asymptotically proportional to the square of the number of steps for long walks; in other words, 〈x2〉=an2, where 〈x2〉 is the mean square x component of net distance traversed and n is the number of steps (assumed to be large). For the two‐layer problem, a=0.5236, and for the three‐layer problem, a=0.3899.

Configurations of macromolecules subject to intermolecular volume exclusions

The effects of intermolecular interactions on the mean square end‐to‐end separations of macromolecules were studied by statistical sampling of multiple nonintersecting random walks on a square planar lattice. The ’’slithering snake’’ procedure was used to obtain large numbers of interacting random configurations, representing chains of 10, 20, 34, 62, and 104 links at various concentrations. The mean square end‐to‐end separation was studied as a function both of overall concentration and of chain length. With increasing concentration, the chains become shorter and the relative shortening becomes more pronounced with increasing contour chain length.

Configurations of macromolecular chains confined to strips or tubes

Theoretical and Monte Carlo studies have been carried out on self‐avoiding random walks confined to relatively narrow strips or tubes. Such walks are used to simulate the behavior of macromolecular chains subject to corresponding external boundary constraints. It is proved that, if a chain is confined to a strip or tube of a width small compared to the contour length of the chain, the mean square end‐to‐end separation of the chain is asymptotically proportional to n2, where n is the number of links in the chain. In addition, Monte Carlo results are reported for strips of three and eight layers and for thin tubes with four and five connected channels. The results so obtained are consistent with the theory. It is conjectured that chains confined to a thin surface, not necessarily monomolecular, will behave as if the chains were limited to a plane, but with parameters different from those for the strictly two‐dimensional problem.