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J Liu, R Bangalore, A Rutledge and DJ Triggle
Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo 14260.
The modulation of L-type Ca2+ channels by membrane depolarization, in terms of channel number, function, and interaction with 1,4- dihydropyridine ligands, has been characterized in clonal rat pituitary cells (GH4C1) and rat cerebellar granule cells. Membrane depolarization by 50 mM extracellular K+ for 120 min caused an approximately 90% reduction in the total number of [3H]PN200-110 binding sites (Bmax) and an approximately 20-fold increase in binding affinity in a whole-cell binding assay. Similar results were obtained in a primary culture of rat cerebellar granule cells. In GH4C1 cells the dissociation constant (Kd) and Bmax were changed from 2.15 nM and 214 fmol/mg at 5 mM K+ to 110 pM and 24 fmol/mg at 50 mM K+, respectively. The changes in affinity and Bmax were both dependent on the extracellular K+ concentration. The affinity change resulted from an increased association rate constant (increased from 0.17 to 3.11 x 10(8) M-1 min- 1 after depolarization) and an unchanged dissociation rate constant (0.032 min-1). Depolarization for 2 hr reduced the number of [3H]PN200- 110 binding sites in the membrane fraction by approximately 50%, but no significant change was detected in total cell homogenates, suggesting removal of L-type Ca2+ channels from the cell surface after depolarization. Blockade of the internalization process by concanavalin A and phenylarsine oxide inhibited the depolarization-induced reduction of L-type Ca2+ channels on the cell surface. A decrease in the number of functional channels on the cell surface, as revealed by stimulated 45Ca2+ uptake, accompanied the change in [3H]PN200-110 binding. Reduction of 45Ca2+ uptake had two exponential components, i.e., rapid (with a time constant of about 2.5 min), with a rapid rate of recovery, and slow (with a time constant of 54 min), with a correspondingly slow rate of recovery. Depolarization of the cells with veratridine (50 microM) or treatment of the cells with the Ca2+ ionophore A23187 (10 microM) had effects similar to those of K+ depolarization on [3H]PN200- 110 binding sites and stimulated 45Ca2+ uptake. The change in [3H]PN200- 110 binding sites in whole-cell and membrane preparations occurred rapidly, becoming prominent within 45 min, and largely recovered when the cells were repolarized. The down-regulation of L-type Ca2+ channels is dependent on Ca2+ entry via a calmodulin-dependent process.
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