BINDING CONSTANTS DETERMINED FROM CA2+ CURRENT RESPONSES TO RAPID APPLICATIONS AND WASHOUTS OF NIFEDIPINE IN FROG CARDIAC MYOCYTES

1. A fast perfusion system was used to analyse the kinetics of the res ponse of L-type calcium current (I-Ca) to rapid applications and washo uts of the dihydropyridine antagonist nifedipine in whole-cell patch-c lamped frog ventricular myocytes. 2. Both the inhibition of I-Ca induc ed by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During applicat ion or washout of 100 nM nifedipine, only the peak amplitude of I-Ca v aried but not its time course of activation or inactivation. 4. The ra te constant of the onset of I-Ca inhibition increased with the concent ration of nifedipine. However, the time course of the recovery from in hibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to t he test potential. 6. Using simple rate equations and a one-binding-si te analysis it was possible to determine the rate constants for associ ation (k(1)) and dissociation (k(-1)) and the equilibrium dissociation constant (K-D) of the reaction between nifedipine and Ca2+ channels. K-D values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potent ials, K-D decreased strongly due to a large reduction in k(-1) and a m odest increase in k(1). Assuming that these changes result from the di stribution of Ca2+ channels between resting and inactivated states: a low-affinity binding to the resting state (R) and a high-affinity bind ing to the inactivated state (I) were obtained with the binding consta nts: k(1)(R) = 1.0 x 10(6) M(-1) s(-1), k(-1)(R) = 0.007 s(-1), and K- D(R) = 77 nM for the resting state; k(1)(I) = 4.47 x 10(6) M(-1) s(-1) , k(-1)(I) = 7.7 x 10(-4) s(-1), and K-D(I) = 0.17 nM for the inactiva ted state. 8. Rapid application/washout experiments provide a unique w ay to determine, in an intact cell and in a relatively short period (2 -4 min), the binding rate constants and the K-D value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.