title: Complex modulation of Ca(v)3.1 T-type calcium channel by nickel.
abstract: Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni(2+)-binding site with critical histidine-191 (H191) within the extracellular IS3-IS4 domain of the most Ni(2+)-sensitive Cav3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni(2+) effect and its dependence on PDC remain uncertain. Here we compared Ni(2+) action on the wild-type "Ni(2+)-insensitive" Cav3.1w/t channel and Cav3.1Q172H mutant having glutamine (Q) equivalent to H191 of Cav3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni(2+) blockade of Ca(2+), Sr(2+), or Ba(2+) currents was assessed by electrophysiology. Inhibition of Cav3.1w/t by Ni(2+) conformed to two sites binding. Ni(2+) binding with high-affinity site (IC50 = 0.03-3 μM depending on PDC) produced maximal inhibition of 20-30% and was voltage-dependent, consistent with its location within the channel's pore. Most of the inhibition (70-80%) was produced by Ni(2+) binding with low-affinity site (IC50 = 240-700 μM). Q172H-mutation mainly affected low-affinity binding (IC50 = 120-160 μM). The IC50 of Ni(2+) binding with both sites in the Cav3.1w/t and Cav3.1Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca(2+), Sr(2+), or Ba(2+) with Ni(2+). We conclude that differential Ni(2+)-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni(2+), may be occupied by PDC, influencing in turn the channel's permeation.
authors.affiliation: International Center of Molecular Physiology of the National Academy of Sciences of Ukraine, Kiev, Ukraine.
authors.forename: Olena V
authors.initials: OV
authors.lastname: Nosal
authors.forename: Olga P
authors.initials: OP
authors.lastname: Lyubanova
authors.forename: Valeri G
authors.initials: VG
authors.lastname: Naidenov
authors.forename: Yaroslav M
authors.initials: YM
authors.lastname: Shuba
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D020747:Calcium Channels, T-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D009532:Nickel / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D014981:Xenopus
chemical_list: D020747:Calcium Channels, T-Type; D002413:Cations, Divalent; D009532:Nickel