Fe²⁺ block and permeation of CaV3.1 (α1G) T-type calcium channels: candidate mechanism for non-transferrin-mediated Fe²⁺ influx

• Published in: Molecular pharmacology 2012-12, Vol.82 (6), p.1194-1204
• Main Authors: Lopin, Kyle V, Gray, I Patrick, Obejero-Paz, Carlos A, Thévenod, Frank, Jones, Stephen W
• Format: Article
• Language: English
• Subjects: Barium - metabolism; Calcium - metabolism; Calcium Channels, T-Type - metabolism; Cell Line; Ferrous Compounds - metabolism; Ferrous Compounds - pharmacology; HEK293 Cells; Humans; Ion Channel Gating - drug effects; Magnesium - metabolism; Membrane Potentials - drug effects; Patch-Clamp Techniques - methods; Transferrin - metabolism
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.112.080184

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe²⁺ on permeation and gating of Ca(V)3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe²⁺ state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe²⁺ rapidly blocked currents with 2 mM extracellular Ca²⁺ in a voltage-dependent manner, as described by a Woodhull model with K(D) = 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe²⁺ also shifted activation to more-depolarized voltages (by ∼10 mV with 1.8 mM extracellular Fe²⁺) somewhat more strongly than did extracellular Ca²⁺ or Mg²⁺, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e(-)/98 Ų and K(Fe) = 4.5 M⁻¹ for extracellular Fe²⁺. In the absence of extracellular Ca²⁺ (and with extracellular Na⁺ replaced by TEA), Fe²⁺ carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at -60 mV with 10 mM extracellular Fe²⁺). With a two-site/three-barrier Eyring model for permeation of Ca(V)3.1 channels, we estimated a transport rate for Fe²⁺ of ∼20 ions/s for each open channel at -60 mV and pH 7.2, with 1 μM extracellular Fe²⁺ (with 2 mM extracellular Ca²⁺). Because Ca(V)3.1 channels exhibit a significant "window current" at that voltage (open probability, ∼1%), Ca(V)3.1 channels represent a likely pathway for Fe²⁺ entry into cells with clinically relevant concentrations of extracellular Fe²⁺.