Multiple types of high-voltage-activated Ca2+ channels, including L-, N-, P-, Q- and R-types have been distinguished from each other mainly employing pharmacological agents that selectively block particular types of Ca2+ channels. Except for the dihydropyridine-sensitive L-type Ca2+ channels, electrophysiological characterization has yet to be conducted thoroughly enough to biophysically distinguish the remaining Ca2+ channel types. In particular, the ion permeation properties of N-type Ca2+ channels have not been clarified, although the kinetic properties of both the L- and N-type Ca2+ channels are relatively well described. To establish ion conducting properties of the N-type Ca2+ channel, we examined a homogeneous population of recombinant N-type Ca2+ channels expressed in baby hamster kidney cells, using a conventional whole cell patch-clamp technique. The recombinant N-type Ca2+ channel, composed of the alpha1B, alpha2a, and beta1a subunits, displayed high-voltage-activated Ba2+ currents elicited by a test pulse more positive than -30 mV, and were strongly blocked by the N-type channel blocker omega-conotoxin-GVIA. In the presence of 110 mM Ba2+, the unitary current showed a slope conductance of 18.2 pS, characteristic of N-type channels. Ca2+ and Sr2+ resulted in smaller ion fluxes than Ba2+, with the ratio 1.0:0. 72:0.75 of maximum conductance in current-voltage relationships of Ba2+, Ca2+, and Sr2+ currents, respectively. In mixtures of Ba2+ and Ca2+, where the Ca2+ concentration was steadily increased in place of Ba2+, with the total concentration of Ba2+ and Ca2+ held constant at 3 mM, the current amplitude went through a clear minimum when 20% of the external Ba2+ was replaced by Ca+2. This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously. By using an external solution containing 110 mM Na+ without polyvalent cations, inward Na+ currents were evoked by test potentials more positive than -50 mV. These currents were activated and inactivated in a kinetic manner similar to that of Ba2+ currents. Application of inorganic Ca2+ antagonists blocked Ba2+ currents through N-type channels in a concentration-dependent manner. The rank order of inhibition was La3+ >/= Cd2+ >> Zn2+ > Ni2+ >/= Co2+. When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La3+ and Cd2+ and subsequent channel reblocking was observed. The measured rate (2 x 10(8) M-1 s-1) of reblocking approached the diffusion-controlled limit. These results suggest that N-type Ca2+ channels share general features of a high affinity ion-binding site with the L-type Ca2+ channel, and that this site is easily accessible from the outside of the channel pore.