EFFECTS OF HEPARIN ON EXCITATION-CONTRACTION COUPLING IN SKELETAL-MUSCLE OF TOAD AND RAT

1. Intracellularly applied heparin was found to cause a novel, use-dependent block of excitation-contraction (E-C) coupling in skinned skeletal muscle fibres of the toad; After one to four depolarizations in the presence of 100 mu g ml(-1) heparin, no further depolarization-induced responses could be elicited, even though addition of caffeine or lowering [Mg2+] could still induce massive Ca2+ release. This effect could not be reversed by extensive wash-out of the heparin (> 15 min). 2. Heparin (100 mu g ml(-1)) did not abolish subsequent depolarization-induced responses if applied while the voltage sensors were in either their resting or inactivated states, that is (a) while a fibre remained fully polarized, (b) when a fibre was already chronically depolarized or (c) after a fibre had been depolarized in the presence of D600 (gallopamil) and then repolarized. 3. When a toad fibre was depolarized in heparin, with the associated Ca2+ release blocked by the presence of 10 mM intracellular Mg2+, subsequent E-C coupling was abolished. Heparin did not interrupt E-C coupling when Ca2+ release was triggered in the absence of any depolarization, by either caffeine or low [Mg2+]. Thus, the opening of the Ca2+ release channels was neither necessary nor sufficient for heparin to abolish E-C coupling. 4. Heparin had direct effects on the contractile apparatus in toad fibres, increasing the Ca2+ sensitivity and decreasing the maximum Ca2+-activated force. These effects could only be partly reversed by extensive wash-out of heparin. 5. At 100 mu g ml(-1), both low molecular weight heparin and pentosanpolysulphate, another highly sulphated polysaccharide, were less effective than heparin in blocking the depolarization-induced response and in changing the properties of the contractile apparatus, and these effects could be substantially reversed by wash-out. Two other polyanions, de-N-sulphated heparin (100 mu g ml(-1)), which lacked N-sulphate groups, and polyglutamate (500 mu g ml(-1)), had no measurable effect on either CC coupling or the contractile apparatus. 6. In skinned fibres of the extensor digitorum longus muscle of the rat, 100 mu g ml-l heparin had little or no effect on E-C coupling and on the Ca2+ sensitivity of the contractile apparatus, but caused a larger reduction of the maximum Ca2+-activated force than in skinned fibres of the toad. 7. These results indicate that heparin blocks E-C coupling in toad muscle if, and only if, it is present when the voltage sensors are activated by depolarization. When the possible heparin binding sites on the voltage sensor/dihydropyridine receptor are considered, these data give further insight into the molecular basis of E-C coupling.