Investigation of pathophysiological properties of ion channels on cardiac sarcoplasmic reticulum.

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(1) Investigation of regulatory mechanism of ryanodine receptor Ca^<2+> release channel. To investigate the regulatory mechanisms of ryanodine receptor Ca^<2+> release channel in cardiac sarcoplasmic reticulum (SR), we purified the SR membrane from pig hearts. These procedures were done in Dr.Coronado's lab at University of Wisconsin. The ryanodine receptors were incorporated into the artificial lipid bilayr and the channel activities were recorded by voltage clamp technique. Dr.Kawano and Dr.Hiraoka visited Dr.Coronado's lab to study and to discuss the procedure of purification of ryanodine receptors from pigs and bovine hearts. By using these preparations, we could record channel activities of ryanodine receptor. In this study we investigated Mg^<2+> effects on this channel and found that Mg^<2+> blocked the channel openings with diverse mechanisms. Namely, (1) Mg^<2+> reduced channel openings by competing with Ca^<2+> at the Ca^<2+> binding activating site of the channel and (2) by … More reducing the channel conductance.Thus, we showed the detail regulatory mechanisms of Ca^<2+> release from SR by Mg^<2+>.(2) Investigation of anion selectivity of a chloride channel in the porcine cardiac sarcoplasmic reticulum. We have reported that Cl-channel are present on cardiac sarcoplasmic reticulum which is activated by cyclic AMP dependent phosphorylation. In this study we examined the ion selectivity of this channel and compare the pore properties of this channel to those of other Cl-channels. Permeability ration calculated by Goldman-Hodgikin-Katz equation revealed the anion permeability sequence as Br->Cl->I->NO_3->F-. Those results were comparable to those of cystic fibrosis transmembrane regulator (CFTR) and the cardiac Cl-channel on the sarcolemma. The biophysical properties of this Cl-channel are very similar to those of the CFTR,suggesting the pore with a moderately high affinity site for anions. Therefor we speculate that cardiac SR Cl-channel may belong to the same family of Cl-channel as CFTR.Dr.Coronado found chloride-induced Ca^<2+> release from sarcoplasmic reticulum. Therefore, we had good discussions about the relationships between these channels on SR and Cl-channel. We could show the new signal tansduction pathway between channels on sarcolemma and those on SR,because of collaboration with Dr.Coronado. Less