Vol. 62, Issue 6, 1418-1430, December 2002

Characteristics of Block by Pb²⁺ of Function of Human Neuronal L-, N-, and R-Type Ca²⁺ Channels Transiently Expressed in Human Embryonic Kidney 293 Cells

Shuangqing Peng, Ravindra K. Hajela, and William D. Atchison

Department of Pharmacology and Toxicology, Institute of Environmental Toxicology and Neuroscience Program, Michigan State University, East Lansing, Michigan

Lead (Pb²⁺) is a well-known inhibitor of voltage-dependent Ca²⁺ channels in their native environments in several types of cells. However, its effects on discrete Ca²⁺ channel phenotypes in isolation have not been well studied. We compared how specific subtypes of human neuronal high-voltage-activated Ca²⁺ channels were affected by acute exposure to Pb²⁺. Expression cDNA clones of human _1C, _1B, or _1E subunit genes encoding neuronal L-, N-, and R-subtypes of Ca²⁺ channels, respectively, along with a constant ₂ and ₃ subunits were transfected into human embryonic kidney 293 cells. Currents through the respective transiently expressed channels were measured using whole-cell recording techniques with Ba²⁺ (20 mM) as charge carrier. Extracellular bath applications of Pb²⁺ significantly reduced current amplitude through all three types of Ca²⁺ channels in a concentration-dependent manner. The order of potency was: _1E (IC₅₀ = 0.10 µM), followed by _1C (IC₅₀ = 0.38 µM) and _1B (IC₅₀ = 1.31 µM). Pb²⁺-induced perturbation of function of _1C and _1B containing Ca²⁺ channels was more easily reversed than for _1E-containing Ca²⁺ channels after washing with Pb²⁺ free solution. The current-voltage relationships were not altered after 3-min exposure to Pb²⁺ for any of the three types. However, the steady-state inactivation relationships were shifted to more negative potentials for channels containing _1B and _1E subunits, but not for those containing _1C subunits. Pb²⁺ accelerated the inactivation time of current in all three subtypes of Ca²⁺ channels in a concentration- and voltage-dependent manner. Therefore, different subtypes of Ca²⁺ channels exhibit differential susceptibility to Pb²⁺ even when expressed in the same cell type. Current expressed by _1E-containing channels is more sensitive to Pb²⁺ than that expressed by _1C- or _1B-containing channels. Several Ca²⁺ channel phenotypes are quite sensitive to the inhibitory action of Pb²⁺. Furthermore, it seems that Pb²⁺ is more likely to combine with Ca²⁺ channels in the closed state.