Functional Characterization of Ion Permeation Pathway in the N-type Ca2+
Channel.
MINORU WAKAMORI, MARK STROBECK, TETSUHIRO NIIDOME, TETSUYUKI TERAMOTO, KEIJI
IMOTO AND YASUO MORI.
Department of Information Physiology, National Institute for Physiological
Sciences, Okazaki, Aichi 444, Japan. Institute of Molecular Pharmacology and
Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH
45267-0828, USA. Eisai Tsukuba Research Laboratories, Tsukuba, Ibaraki 300-26,
Japan.
APStracts 4:308N, 1997.
ABSTRACT
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 a1B, a2a and b1a 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 w-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 Ca2+. This anomalous mole
fraction effect suggests an ion-binding site where two or more permeant ions
can sit simultaneously. 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
108 M-1s-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.
Received 29 May 1997; accepted in final form 31 October 1997.
APS Manuscript Number J445-7.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1997 The American Physiological Society.
Published in APStracts on 14 November 1997