The effect of phenylglyoxal on contraction and intramembrane charge movement in frog skeletal muscle.

Abstract

1. The effects of the arginine-specific protein-modifying reagent, phenylglyoxal, on contraction and intramembrane charge movement were studied in cut single fibres from frog skeletal muscle, using the double-Vaseline-gap voltage clamp technique. 2. The strength-duration curve for pulses which produced microscopically just-detectable contractions was shifted to more positive potentials and longer durations following treatment of fibres with phenylglyoxal. Caffeine-induced contractures were not blocked. 3. The amount of charge moved by large depolarizing pulses from -100 mV holding potential (charge 1) declined during the phenylglyoxal treatment with a single-exponential time course (tau = 7 min). Linear capacitance did not change significantly over the entire experiment. Inhibition of charge movement was predominantly irreversible. 4. Slow bumps (Q gamma) observed in charge movement current transients recorded before phenylglyoxal treatment, using either large test pulses or small steps superimposed on test pulses, were absent from currents recorded after treatment. The current removed by phenylglyoxal contained the bump (Q gamma) and a small fast transient (Q beta). 5. The amount of charge moved by large depolarizing pulses from -100 mV was reduced 20-50% following phenylglyoxal treatment. Charge moved by pulses to potentials more negative than -40 mV was relatively unaffected. The magnitude and voltage range of this inhibitory effect were the same whether the reagent was applied at -100 mV or at 0 mV holding potential. 6. A phenylglyoxal-sensitive component of charge was isolated which had a much steeper voltage dependence than the total charge movement or the charge remaining after treatment. 7. Charge recorded during hyperpolarizing pulses from 0 mV holding potential (charge 2) was reduced very little (less than 5%) at any potential by phenylglyoxal treatments at either 0 or -100 mV. 8. The phenylglyoxal reaction with charge 2 was kinetically different from the reaction with charge 1. 9. The effects of phenylglyoxal on charge 1 and charge 2 both measured in the same fibre were compared. Whether phenylglyoxal was applied at -100 mV, or at 0 mV the results were the same: charge 1 was inhibited much more (25-60%) than charge 2(2-12%). 10. The results presented here indicate that arginyl residues have a functional role in the voltage-sensing mechanism of excitation-contraction coupling and support the hypothesis that the dihydropyridine receptor is the voltage sensor molecule.