The Journal of Neurophysiology Vol. 79 No. 2 February 1998, pp. 622-634
Copyright ©1998 The American Physiological Society

Functional Characterization of Ion Permeation Pathway in the N-Type Ca²⁺ Channel

Minoru Wakamori^{1, 2}, Mark Strobeck^{1, 2}, Tetsuhiro Niidome³, Tetsuyuki Teramoto³, KEIJI Imoto¹, and Yasuo Mori^{1, 2}

¹ Department of Information Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444, Japan; ² Institute of Molecular Pharmacology and Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0828; and ³ Eisai Tsukuba Research Laboratories, Tsukuba, Ibaraki 300-26, Japan

Wakamori, Minoru, Mark Strobeck, Tetsuhiro Niidome, Tetsuyuki Teramoto, Keiji Imoto, and Yasuo Mori. Functional characterization of ion permeation pathway in the N-type Ca²⁺ channel. J. Neurophysiol. 79: 622-634, 1998. 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 _1B, _2a, and _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 -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 × 10⁸ M¹ s¹) 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.

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