J. Biol. Chem., Vol. 263, Issue 21, 10155-10161, 07, 1988
Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings
SE Guggino, JA Wagner, AM Snowman, LD Hester, B Sacktor and SH Snyder
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
(-)-[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.
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