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

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 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.

The Role of a Voltage-Dependent Ca2+Channel Intracellular Linker: A Structure-Function Analysis

“…6C). This phenomenon was previously reported in Ca V 2.2 studies but its origin is not completely understood (Fox et al, 1987;Jones and Marks, 1989;Patil et al, 1998;Wakamori et al, 1998;Wakamori et al, 1999;Kang et al, 2001;Meuth et al, 2002;Tselnicker et al, 2010). Furthermore, our PL mutations seem to affect this behavior and replacing the PL (Ca V 2.2/Ca V 1.2 PL) seems to have eliminated it (Fig.…”

Single Tottering Mutations Responsible for the Neuropathic Phenotype of the P-type Calcium Channel

“…Rapid application of drugs were made by a modified "Y-tube" method. Details of this technique have already appeared (30). The external solution surrounding a cell recorded was completely exchanged within 200 ms. Statistical comparison between normal and mutant mice or mutant channels was performed by Student's t test (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001).…”

Aspartate Residues of the Glu-Glu-Asp-Asp (EEDD) Pore Locus Control Selectivity and Permeation of the T-type Ca2+Channel α1G

“…The most representative is perhaps that the Ba 2ϩ conductance of most HVA Ca 2ϩ channels is ϳ2 times larger than for Ca 2ϩ (1)(2)(3), whereas the Ca 2ϩ conductance of LVA Ca 2ϩ channels is identical to or larger than that of Ba 2ϩ (4 -6). In some experimental conditions, Ba 2ϩ currents are blocked by Ca 2ϩ in HVA channels, a phenomenon known as anomalous mole fraction effect (AMFE) that is a result of the higher binding affinity of these channels for Ca 2ϩ (7)(8)(9)(10)(11)(12).…”