Characterisation of the divalent cation channels of the hepatocyte plasma membrane receptor-activated Ca²⁺ inflow system using lanthanide ions

The ability of Gd³⁺ to inhibit vasopressin-stimulated Ca²⁺ inflow to hepatocytes was compared with its effect on Mn²⁺ inflow. In the absence of Gd³⁺, the stimulation of Mn²⁺ inflow by vasopressin increased with increasing pH of the extracellular medium. Maximal inhibition of vasopressin-stimulated Ca²⁺ and Mn²⁺ inflow by saturating concentrations of Gd³⁺ was 70 and 30%, respectively. Gd³⁺ also inhibited thapsigargin-stimulated Ca²⁺ and Mn²⁺ inflow with maximal inhibition of 70 and 40%, respectively. It is concluded that vasopressin and thapsigargin each activate two types of Ca²⁺ inflow processes, one which is sensitive and one which is insensitive to lanthanides. The nature of the pore of the lanthanide-sensitive Ca²⁺ channel was investigated further using different lanthanides as inhibitors. Tm³⁺, Gd³⁺, Eu³⁺, Nd³⁺ and La³⁺ each inhibited vasopressin-stimulated Ca²⁺ and Mn²⁺ inflow but had no effect on Ca²⁺ inflow in the absence of an agonist, or on vasopressin-stimulated release of Ca²⁺ from intracellular stores. Maximal inhibition of vasopressin-stimulated Ca²⁺ inflow in the presence of a saturating concentration of each lanthanide ranged from 70-90%. An equation which describes a 1:1 interaction of the lanthanide with a putative binding site in the Ca²⁺ channel gave a good fit to dose-response curves for the inhibition of vasopressin-stimulated Ca²⁺ inflow by each lanthanide. Lanthanides in the middle of the series exhibited the lowest dissociation constant (K_d) values. The K_d for Gd³⁺ increased with increasing extracellular Ca²⁺ concentration, suggesting competitive inhibition of Ca²⁺ binding by Gd³⁺. In the absence of lanthanide, vasopressin-stimulated Mn²⁺ inflow was substantially reduced when the plasma membrane was depolarised by increasing the extracellular K⁺ concentration. Changing the membrane potential had little effect on the maximum inhibition by Gd³⁺ of vasopressin-stimulated Mn²⁺ inflow. The K_d for inhibition of vasopressin-stimulated Ca²⁺ inflow by Gd³⁺, measured at the lowest attainable membrane potential, was about 6-fold lower than the K_d measured at the highest attainable membrane potential. The idea that there is a site in the vasopressin-stimulated lanthanide-sensitive Ca²⁺ channel composed of carboxylic acid groups which bind Ca²⁺, Mn²⁺ or a lanthanide ion is consistent with the data obtained using the different lanthanides.