MODULATION OF L-TYPE CA2-CELLS BY MEMBRANE DEPOLARIZATION( CHANNELS IN CLONAL RAT PITUITARY)

The modulation of L-type Ca2+ channels by membrane depolarization, in terms of channel number, function, and interaction with 1,4-dihydropyr idine ligands, has been characterized in clonal rat pituitary cells (G H(4)C(1)) and rat cerebellar granule cells. Membrane depolarization by 50 mM extracellular K+ for 120 min caused an approximately 90% reduct ion in the total number of [H-3]PN200-110 binding sites (B-max) and an approximately 20-fold increase in binding affinity in a whole-cell bi nding assay. Similar results were obtained in a primary culture of rat cerebellar granule cells. In GH(4)C(1) cells the dissociation constan t (K-d) and B-max 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 af finity and B-max were both dependent on the extracellular K+ concentra tion. 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 depo larization) and an unchanged dissociation rate constant (0.032 min(-1) ). Depolarization for 2 hr reduced the number of [H-3]PN200-110 bindin g sites in the membrane fraction by approximately 50%, but no signific ant change was detected in total cell homogenates, suggesting removal of L-type Ca2+ channels from the cell surface after depolarization. Bl ockade of the internalization process by concanavalin A and phenylarsi ne 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 Ca-45(2+) upta ke, accompanied the change in [H-3]PN200-110 binding. Reduction of Ca- 45(2+) 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 r ecovery. Depolarization of the cells with veratridine (50 mu M) or tre atment of the cells with the Ca2+ ionophore A23187 (10 mu M) had effec ts similar to those of K+ depolarization on [H-3]PN200-110 binding sit es and stimulated Ca-45(2+) uptake. The change in [H-3]PN200-110 bindi ng sites in whole-cell and membrane preparations occurred rapidly, bec oming prominent within 45 min, and largely recovered when the cells we re repolarized. The down-regulation of L-type Ca2+ channels is depende nt on Ca2+ entry via a calmodulin-dependent process.