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 (Kd) values. The Kd 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 Kd for inhibition of vasopressin-stimulated Ca2+ inflow by Gd3+, measured at the lowest attainable membrane potential, was about 6-fold lower than the Kd 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.
Fernando
K C
KC
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Barritt
G J
GJ
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000187:drug effects*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008564:Membrane Potentials; D008674:Metals, Rare Earth / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D051381:Rats; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014667:Vasopressins / Q000494:pharmacology
D015220:Calcium Channels; D002412:Cations; D007473:Ion Channels; D008674:Metals, Rare Earth; D013729:Terpenes; D014667:Vasopressins; D008345:Manganese; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D011188:Potassium; D002118:Calcium