KINETIC ISOFORMS OF INTRAMEMBRANE CHARGE IN INTACT AMPHIBIAN STRIATED-MUSCLE

The effects of the ryanodine receptor (RyR) antagonists ryanodine and daunorubicin on the kinetic and steady-state properties of intramembra ne charge were investigated in intact voltage-clamped frog skeletal mu scle fibers under conditions that minimized time-dependent ionic curre nts. A hypothesis that RyR gating is allosterically coupled to configu rational changes in dihydropyridine receptors (DHPRs) would predict th at such interactions are reciprocal and that RyR modification should i nfluence intramembrane charge. Both agents indeed modified the time co urse of charging transients at 100-200-mu M concentrations. They indep endently abolished the delayed charging phases shown by currents, even in fibers held at fully polarized, -90-mV holding potentials; such wa veforms are especially prominent in extracellular solutions containing gluconate. Charge movements consistently became exponential decays to stable baselines in the absence of intervening inward or other time-d ependent currents. The steady-state charge transfers nevertheless rema ined equal through the ON and the OFF parts of test voltage steps. The charge-voltage function, Q(V-T), shifted by similar to+10 mV, particu larly through those test potentials at which delayed q(gamma) currents normally took place but retained steepness factors (k approximate to 8.0 to 10.6 mV) that indicated persistent, steeply voltage-dependent q (gamma) contributions. Furthermore, both RyR antagonists preserved the total charge, and its variation with holding potential, Q(max)(V-H), which also retained similarly high voltage sensitivities (k approximat e to 7.0 to 9.0 mV). RyR antagonists also preserved the separate ident ities of q(gamma) and q(beta) species, whether defined by their steady -state voltage dependence or inactivation or pharmacological propertie s. Thus, tetracaine (2 mM) reduced the available steady-state charge m ovement and gave shallow Q(V-T) (k approximate to 14 to 16 mV) and Q(m ax)(V-H) (k approximate to 14 to 17 mV) curves characteristic of q(bet a) charge. These features persisted with exposure to test agent. Final ly, q(gamma) charge movements showed steep voltage dependences with bo th activation (k approximate to 4.0 to 6.5 mV) and inactivation charac teristics (k approximate to 4.3 to 6.6 mV) distinct from those shown b y the remaining q(beta) charge, whether isolated through differential tetracaine sensitivities, or the full approximation of charge-voltage data to the sum of two Boltzmann distributions. RyR modification thus specifically alters q(gamma) kinetics while preserving the separate id entities of steady-state q(beta) and q(gamma) charge. These findings p ermit a mechanism by which transverse tubular voltage provides the pri mary driving force for configurational changes in DHPRs, which might p roduce q(gamma) charge movement. However, they attribute its kinetic c omplexities to the reciprocal allosteric coupling by which DHPR voltag e sensors and RyR-Ca2+ release channels might interact even though the se receptors reside in electrically distinct membranes. RyR modificati on then would still permit tubular voltage change to drive net q(gamma ) charge transfer but would transform its complex waveforms into simpl e exponential decays.