Functional characterization of ion permeation pathway in the N-type Ca²⁺ channel

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 α(1B), α(2a), and β(1a) 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 ω-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 C²⁺ 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 Ca²⁺. 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⁺ antagonism 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 10⁸ 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.