Vol. 289, Issue 1, 486-493, April 1999

Mechanisms of -Hexachlorocyclohexane Toxicity: II. Evidence for Ca²⁺-Dependent K⁺-Selective Ionophore Activity¹

Edmond D. Buck and Isaac N. Pessah

Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California

-Hexachlorocyclohexane (-HCH) interacts with cardiac ryanodine-sensitive Ca²⁺ channels (RyR2), accounting in part for altered Ca²⁺ transients and contractility (reported in companion report). Analysis of channel gating kinetics in the presence of -HCH also revealed a nonfluctuating membrane current that remained even after RyR2 channels were blocked. We further elucidated the nature of a direct interaction between -HCH and biological membranes by measuring ionic currents across planar lipid bilayers made from defined lipids lacking cellular protein using voltage-clamp. Dimethyl sulfoxide, in the presence or absence of 50 µM -HCH (lindane) or -HCH, produced negligible steady-state current with symmetric 100 mM CsCl in the range of ±50 mV. However, the addition of 50 µM Ca²⁺ to the bilayer chamber in the presence of -HCH induced a profound increase in ionic permeability that was not seen in the presence of -HCH or dimethyl sulfoxide control. Significantly, the permeability increase 1) was proportional with increasing Ca²⁺ to ~600 µM and saturated between 1 and 2 mM Ca²⁺ regardless of holding potential, 2) occurred only when -HCH and Ca²⁺ were added to the same side of the membrane, and 3) was independent of the order of addition or of the side of the membrane to which -HCH and Ca²⁺ was added. The Ca²⁺-dependent current produced by -HCH was highly selective for monovalent cations (K⁺ Cs⁺ > Na⁺), with negligible conductance for Ca²⁺ or Cl. In symmetric 100 mM K⁺, the conductance induced with 50 µM concentration each of -HCH and Ca²⁺ was 4.25 pA/mV. The results show that -HCH increases the ionic permeability of phospholipid membranes by two distinct Ca²⁺-dependent mechanisms: one mediated through RyR and the other mediated by a unique ionophore activity.