SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has 
recently been described as a potent and selective inhibitor of Na(+)-Ca(2+) 
exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of 
SEA0400 were investigated on the cloned cardiac Na(+)-Ca(2+) exchanger, NCX1.1, 
expressed in Xenopus laevis oocytes to gain insight into its inhibitory 
mechanism. Na(+)-Ca(2+) exchange currents were measured using the giant excised 
patch technique using conditions to evaluate both inward and outward currents. 
SEA0400 inhibited outward Na(+)-Ca(2+) exchange currents with high affinity 
(IC(50) = 78 +/- 15 and 23 +/- 4 nM for peak and steady-state currents, 
respectively). Considerably less inhibitory potency (i.e., micromolar) was 
observed for inward currents. The inhibitory profile was reexamined after 
proteolytic treatment of excised patches with alpha-chymotrypsin, a procedure 
that eliminates ionic regulatory mechanisms. After this treatment, an IC(50) 
value of 1.2 +/- 0.6 microM was estimated for outward currents, whereas inward 
currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400 
on outward exchange currents were evident at both high and low concentrations of 
regulatory Ca(2+), although distinct features were noted. SEA0400 accelerated 
the inactivation rate of outward currents. Based on paired pulse experiments, 
SEA0400 altered the recovery of exchangers from the Na(+)(i)-dependent inactive 
state, particularly at higher regulatory Ca(2+)(i) concentrations. Finally, the 
inhibitory potency of SEA0400 was strongly dependent on the intracellular Na(+) 
concentration. Our data confirm that SEA0400 is the most potent inhibitor of the 
cardiac Na(+)-Ca(2+) exchanger described to date and provide a reasonable 
explanation for its apparent transport mode selectivity.