• The organization of beta-spectrin and dystrophin at the sarcolemma of skeletal muscle

      Williams, McRae Witherspoon; Bloch, Robert J. (1998)
      Dystrophin, the protein missing in Duchennes muscular dystrophy, is structurally similar to, beta-spectrin. Immunofluorescence studies have shown that these two proteins have similar distributions within skeletal muscle fibers. I use immunofluorescence and immunoprecipitation to examine beta-spectrin and dystrophin in innervated and denervated fast and slow twitch muscle, and to determine how beta-spectrin is anchored to the membrane. I also examine the organization of the sarcolemma in mdx mice, which lack dystrophin, and in ja/ja mice which lack beta-spectrin. My results indicate that beta-spectrin and dystrophin colocalize at most areas of the sarcolemma in skeletal muscle, but that in fast twitch fibers, "intracostameric regions" and "juxtanuclear domains" contain dystrophin but little or no beta-spectrin. This indicates that the distribution of dystrophin at the sarcolemma is not dependent on beta-spectrin, and suggests that broad regions of the sarcolemma are likely to be poorly supported in the absence of dystrophin. I demonstrate that the distributions of both beta-spectrin and dystrophin are different in fast and slow twitch fibers, and that this distribution is affected by denervation. Denervating both fast and slow twitch fibers causes both proteins to redistribute in a pattern resembling that seen in healthy slow twitch fibers. This indicates that the distribution of these proteins is influenced by innervation. I determine that beta-spectrin is anchored to the membrane through its association with the alpha-subunit of the Na+/K+ ATPase. In ja/ja mice, the Na+/K+ ATPase distributes more uniformly in the sarcolemma. Dystrophin is believed to connect the membrane to the contractile apparatus through its interaction with actin and its membrane receptor, the dystrophin-associated glycoprotein complex. Through its ability to interact with the Na+/K+ ATPase, beta-spectrin, may serve a similar role. Finally I show that in mdx mice, beta-spectrin distributes abnormally. Other membrane-associated proteins like beta-dystroglycan, syntrophin, and the Na+/K+ ATPase distribute abnormally together with beta-spectrin. This suggests that the normal distribution of beta-spectrin and other proteins of the cytoskeleton may depend on dystrophin. It further suggests that the spectrin-based membrane skeleton and the dystrophin-based membrane skeleton in skeletal muscle are linked by a protein other than dystrophin itself.
    • Sodium-calcium exchange current in giant excised patches of cardiac sarcolemma: Characteristics of proton block

      Doering, Andrea Elaine; Lederer, W. Jonathan (1992)
      Sodium-calcium exchange current was recorded under voltage clamp in giant excised sarcolemmal patches from adult guinea pig ventricular myocytes. An outward sodium-calcium exchange current was activated by a step increase in cytoplasmic sodium. The sodium-activated current showed a sigmoid dependence on cytoplasmic sodium concentration and a biphasic dependence on cytoplasmic calcium concentration, with peak current amplitude at 1 {dollar}\mu{dollar}M calcium. The sodium-activated current was inhibited by lanthanum, nickel, amiloride, and protons. These characteristics agree with measurements of sodium-calcium exchange in other preparations. The data suggested that block by lanthanum and cobalt is enhanced by membrane depolarization. The sodium-calcium exchange current was highly sensitive to cytoplasmic pH above 6.0. At 6.0 it was completely blocked. Proton block was not relieved by increased cytoplasmic sodium, but did appear to be relieved by increased cytoplasmic calcium, suggesting that protons compete at a calcium binding site. The potency of proton block was not measurably reduced by a temperature drop from 34{dollar}\sp\circ{dollar}C to 22{dollar}\sp\circ{dollar}C, as if proton interaction with the sodium-calcium exchanger involves a simple physical reaction. Partial proteolysis of the sodium-calcium exchanger with {dollar}\alpha{dollar}-chymotrypsin reduced its sensitivity to proton block. Proton block develops in two phases: the first phase was complete in less than one second and occurred in the absence of cytoplasmic sodium, and the second phase developed with an average half time of five seconds and did not occur in the absence of cytoplasmic sodium. Proton inhibition of sodium-calcium exchange was modeled as a series of first- and second-order reactions, such that the sodium-bound form of the sodium-calcium exchanger has a higher affinity for protons than the sodium-free form. This model reproduced the observed sensitivity of proton block of sodium-calcium exchange current to cytoplasmic sodium. In conclusion, protons inhibit the cardiac sodium-calcium exchanger by multiple mechanisms, at least one of which may involve competition at a calcium binding site. Proton block is potentiated by increased cytoplasmic sodium, which means that a rise in cytoplasmic sodium concentration will cause a fast activation of sodium-calcium exchange followed by a slow development of proton block, even if pH remains constant.