The sarcoplasmic reticulum (SR) is an intracellular membrane system dedicated to the active regulation of cytosolic calcium in muscle. The opening of Ca²⁺ channels in the SR results in a rapid increase in the myoplasmic Ca²⁺ concentration and the initiation of contraction. Closure of these channels allows the SR to re-accumulate the released Ca²⁺ which results in muscle relaxation. While it is known that a muscle fiber is stimulated to contract by the depolarization of the sarcolemma, it is not understood how this signal is communicated to the SR. The focus of this dissertation is twofold. The first objective is to gain an understanding of the mechanism of Ca²⁺ release from the SR. To this end, three studies have been performed which indicate that Ca²⁺ release is mediated by an oxidation reaction. The second goal is to gain insight into the function of the Ca²⁺ release channel. This is addressed by a fourth study which characterizes the effect of the plant alkaloid, ryanodine on channel operation. The anthraquinones mitoxantrone , doxorubicin, daunorubicin, and rubidazone are shown to be potent stimulators of Ca²⁺ release from SR vesicles. Anthraquinoneinduced Ca²⁺ release is shown to be via a specific interaction with the Ca²⁺ release system of the SR. In addition, a strong interaction between anthraquinone and caffeine binding sites on the Ca²⁺ release channel is observed when monitoring Ca²⁺ fluxes across the SR. It is shown that Ca²⁺ release stimulated by anthraquinones is inhibited by preincubating the quinone with dithionite, a strong reducing agent. Spectrophotometric measurements show that the dithionite treated quinone is in a reduced state. Previous work in this lab has shown that the photooxidizing xanthene dye rose bengal stimulates rapid Ca²⁺ release from skeletal muscle SR vesicles. In this thesis, it is shown that following fusion of vesicles to a bilayer lipid membrane (BLM), Ca²⁺ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. This stimulation is shown to be independent of the Ca²⁺ concentration but is inhibited by μM ruthenium red. The photooxidation of rose bengal is shown to not affect either the K+ or Cl- channels which are present in the SR. Exposure of the Ca²⁺ release channel to 500 nM rose bengal in the presence of light is shown to reverse the modification to the channel induced by μM ryanodine. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is directly observed upon measurement of [³H]ryanodine binding to TSR vesicles. Evidence is presented which suggests that Ca²⁺ release is mediated by singlet oxygen. Micromolar concentrations of the porphyrin meso-Tetra(4-N-methylpyridyl)porphine tetraiodide (TMPyP) is shown to induce the rapid release of Ca²⁺ from skeletal muscle SR vesicles. Porphyrin-induced Ca²⁺ release is stimulated by adenine nucleotides and μM Ca²⁺, and is inhibited by mM Mg²⁺ and μM ruthenium red. High-affinity [³H]ryanodine binding is also enhanced in the presence of the porphyrin. The presence of 1 mM Mg²⁺ in the assay medium sensitizes ryanodine binding to activation by ca²⁺. Porphyrin stimulated single channel activity is also sensitized to activation by Ca²⁺ in the presence of Mg²⁺. Reduction of the porphyrin by dithionite, a strong reducing agent, prior to exposure to the Ca²⁺ release channel inhibited the ability of TMPyP to stimulate Ca²⁺ release. These observations indicate that anthraquinones, rose bengal , and porphyrins induce a stimulation of the Ca²⁺ release protein from skeletal muscle SR by interacting with the ryanodine binding site. In addition, the mechanism of interaction for these compounds appears to be via an oxidation reaction. Nanomolar to micromolar concentrations of ryanodine are shown to alter the gating kinetics of the Ca²⁺ release channel from skeletal muscle SR fused with bilayer lipid membranes. In the presence of asymmetric CsCl, 5 to 40 nM concentrations of ryanodine are shown to activate the channel by increasing the open probability (P₀) without changing the conductance. Statistical analysis of gating kinetics reveal that the open and closed dwell times exhibit bi-exponential distributions that are significantly modified by nM ryanodine. The altered channel gating kinetics seen with low nM ryanodine is reversible and is shown to correlate with the binding kinetics of [³H]ryanodine with its highest affinity site under identical ionic conditions. Ryanodine concentrations between 20 and 50 nM are observed to induce occasional 1/2 conductance fluctuations while ryanodine concentrations greater than 50 nM stabilize the channel into a ½ conductance state which is not reversible. These results are shown to correlate with [³H]ryanodine binding to a second site having lower affinity than the first site. Ryanodine at concentrations greater than 70 μM from the 1/2 to a 1/4 conductance fluctuation , whereas ryanodine concentrations greater than 200 μM cause complete closure of the channel. The concentration of ryanodine required to stabilize either the 1/4 conductance transitions or channel closure do not directly correlate with the measured [³H]ryanodine equilibrium binding constants. However, these results can be explained by considering the association kinetics of ryanodine concentrations greater than 200 nM in the presence of 500 mM CsCl. These results indicate that ryanodine stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting ryanodine binding sites on the channel protein.

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published_or_final_version / Zoology / Master / Master of Science

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