Identification and properties of voltage-sensitive sodium channels in smooth muscle cells from pregnant rat myometrium.
Saturable high and low affinity binding sites for [3H]saxitoxin were identified in myometrial membranes of pregnant rats, with dissociation constants of 0.53 and 27 nM, respectively. The maximal binding capacity of the low affinity binding sites was about 10 times higher than that of the high affinity binding sites. The dissociation constants obtained from association and dissociation kinetics of [3H]saxitoxin were similar to those obtained from equilibrium binding. Saxitoxin and tetrodotoxin specifically displaced [3H]saxitoxin binding at both types of sites. Isradipine (1-10 microM) and amiloride (50-100 microM) were without effect on the binding of [3H]saxitoxin. At high concentrations (10-100 microM), veratridine induced a partial inhibition of [3H]saxitoxin binding. In dispersed myometrial cells, [3H]saxitoxin binding revealed the presence of both high and low affinity binding sites, with KD values similar to those obtained in myometrial membranes. Sodium currents were studied in both freshly dispersed and cultured myometrial cells in the presence of veratridine (100 microM), using the whole-cell patch-clamp technique. Steady state inactivation curves indicated that sodium channels were available at negative membrane potentials (between -110 and -40 mV). Isradipine (1-10 microM) and amiloride (50-100 microM) were without effect on the sodium current. Applications of saxitoxin or tetrodotoxin inhibited the amplitude of the sodium current in a concentration-dependent manner. The concentrations of saxitoxin and tetrodotoxin producing half-maximal inhibition were 1.4 and 8.8 nM, respectively. Although the IC50 values for saxitoxin and tetrodotoxin found from electrophysiological experiments are not identical to the equilibrium dissociation constants for the high and low affinity sites found from binding experiments, these results suggested that binding of the neurotoxins to the high affinity sites may be involved in their inhibitory effects on sodium channels. Furthermore, low affinity binding sites may be associated with a non-functional subtype of sodium channels in myometrial cells.
Martin
C
C
Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, INSERM CJF 88-13, Université de Bordeaux II, France.
Arnaudeau
S
S
Jmari
K
K
Rakotoarisoa
L
L
Sayet
I
I
Dacquet
C
C
Mironneau
C
C
Mironneau
J
J
D000200:Action Potentials; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D004553:Electric Conductivity / Q000187:drug effects; D005260:Female; D017275:Isradipine; D009130:Muscle, Smooth / Q000378:metabolism*; D009215:Myometrium / Q000737:chemistry; D011247:Pregnancy; D011725:Pyridines / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D012530:Saxitoxin / Q000378:metabolism / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
D011725:Pyridines; D015222:Sodium Channels; D012530:Saxitoxin; D013779:Tetrodotoxin; D014701:Veratridine; D000584:Amiloride; D017275:Isradipine