Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings.
(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.
Guggino
S E
SE
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Wagner
J A
JA
Snowman
A M
AM
Hester
L D
LD
Sacktor
B
B
Snyder
S H
SH
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002460:Cell Line; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D010006:Osteoblasts / Q000502:physiology*; D012516:Osteosarcoma / Q000503:physiopathology*; D011978:Receptors, Nicotinic / Q000378:metabolism; D014700:Verapamil / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002413:Cations, Divalent; D002414:Cations, Monovalent; D007473:Ion Channels; D011978:Receptors, Nicotinic; C043184:4-desmethoxyverapamil; D014700:Verapamil; D002118:Calcium