EFFECTS OF PERCHLORATE ON THE MOLECULES OF EXCITATION-CONTRACTION COUPLING OF SKELETAL AND CARDIAC-MUSCLE

To understand the nature of the transmission process of excitation-con traction (EC) coupling, the effects of the anion perchlorate were inve stigated on the voltage sensor (dihydropyridine receptor, DHPR) and th e Ca release channel (ryanodine receptor, RyR) of the sarcoplasmic ret iculum (SR). The molecules, from rabbit skeletal muscle, were either s eparated in membrane vesicular fractions or biochemically purified so that the normal EC coupling interaction was prevented. Additionally, t he effect of ClO4- was investigated on L-type Ca2+ channel gating curr ents of guinea pig ventricular myocytes, as a native DHPR not in the p hysiological interaction of skeletal muscle. At 20 mM, ClO4- had minor effects on the activation of ionic currents through Ca channels from skeletal muscle transverse tubular (T) membranes fused with planar bil ayers: a +7-mV shift in the midpoint voltage, VBAR, with no change in kinetics of activation or deactivation. This is in contrast with the l arger, negative shift that ClO4- causes on the distribution of intrame mbrane charge movement of skeletal muscle. At up to 100 mM it did not affect the binding of the DHP [H-3]PN200-110 to triad-enriched membran e fractions (TR). At 8 mM it did not affect the kinetics or the voltag e distribution of gating currents of Ca channels in heart myocytes. Th ese negative results were in contrast to the effects of ClO4- on the r elease channel. At 20 mM it increased several-fold the open probabilit y of channels from purified RyR incorporated in planar bilayers and co nducting Ba2+, an effect seen on channels first closed by chelation of Ca2+ or by the presence of Mg2+. It significantly increased the initi al rate of efflux of Ca-45(2+) from TR vesicles (by a factor of 1.75 a t 20 mM and 4.5 at 100 mM). ClO4- also increased the binding of [H-3]r yanodine to TR fractions. The relative increase in binding was 50-fold at the lowest [Ca2+] used (1 muM) and then decayed to much lower valu es as [Ca2+] was increased. The increase was due entirely to an increa se in the association rate constant of ryanodine binding. The chaotrop ic ions SCN- and I- increased the association rate constant to a simil ar extent. The binding of ryanodine to purified RyR protein reconstitu ted into liposomes had a greater affinity than to TR fractions but was similarly enhanced by ClO4-. The reducing agent dithiothreitol (5 mM) did not reduce the effect of ClO4-, and 5% polyethylene glycol, with an osmolarity equivalent to 20 mM ClO4-, did not change ryanodine bind ing. The results are consistent with a primary effect of ClO4- on the release channel and suggest that the effect on charge movement may be secondary, mediated by a mechanical interaction between the voltage se nsor and the release channel.