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 response of L-type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole-cell patch-clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one-binding-site analysis it was possible to determine the rate constants for association (k1) and dissociation (k-1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k-1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low-affinity binding to the resting state (R) and a high-affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M-1 S-1, k-1R = 0.077 S-1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M-1 S-1, k-1I = 7.7 x 10(-4) S-1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2-4 min), the binding rate constants and the KD value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.
Méry
P F
PF
Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université de Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, France.
Hove-Madsen
L
L
Mazet
J L
JL
Hanf
R
R
Fischmeister
R
R
D000818:Animals; D015220:Calcium Channels / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006321:Heart / Q000187:drug effects*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects*; D009543:Nifedipine / Q000494:pharmacology*; D011893:Rana esculenta
D015220:Calcium Channels; D009543:Nifedipine