guanosine-diphosphate and 1-4-dihydropyridine

Frequent strong depolarizations facilitate Ca2+ channels in various cell types by shifting their gating behavior towards mode 2, which is characterized by long openings and high probability of being open. In cardiac cells, the same type of gating behavior is potentiated by beta-adrenoceptors presumably acting via phosphorylation of a protein identical to or associated with the channel. Voltage-dependent phosphorylation has also been reported to underlie Ca2+ channel facilitation in chromaffin adrenal medulla and in skeletal muscle cells. We studied a possible voltage-dependent facilitation of the principal channel forming alpha 1-subunit of the dihydropyridine-sensitive smooth muscle Ca2+ channel. Single channel and whole-cell Ca2+ currents were recorded in Chinese hamster ovary cells stably expressing the class Cb Ca2+ channel alpha 1-subunit. Strong depolarizing voltage-clamp steps preceding the test pulse resulted in a 2- to 3-fold increase of the single Ca2+ channel activity and induction of mode 2-like gating behavior. Accordingly we observed a significant potentiation of the whole-cell current by approximately 50%. In contrast to the previous suggestions we found no experimental evidence for involvement of channel phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C and other protein kinases utilizing ATP gamma S) in the control and facilitated current. The data demonstrate that the L-type Ca2+ channel alpha 1-subunit solely expressed in Chinese hamster ovary cells is subject to a voltage-dependent facilitation but not to phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Carrier Proteins; CHO Cells; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Dihydropyridines; DNA, Complementary; Enzyme Inhibitors; GTP-Binding Proteins; Guanosine Diphosphate; Intracellular Signaling Peptides and Proteins; Ion Channel Gating; Membrane Potentials; Muscle, Smooth; Phosphorylation; Protein Kinase C; Thionucleotides

Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110 to membrane preparations from rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites (Kd = 0.30 +/- 0.05 nM) that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]-PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 microM for the agonist (+/-)-Bay K8644 and for the antagonists nifedipine, (+/-)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since (-)-PN200-110 (1-200 microM) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) on binding parameters. At a concentration of 10 microM, neither GTP gamma S nor GDP beta S significantly affected the binding of dihydropyridines to their high-affinity sites. GTP gamma S did, however, increase the ability of (+/-)-Bay K8644, but not of (+/-)-nitrendipine, to accelerate the rate of dissociation of tightly bound (+)-[3H]PN200-110. GDP beta S did not affect the dose dependence of either the agonist or the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Isradipine; Kinetics; Ligands; Membrane Proteins; Microsomes; Muscles; Nitrendipine; Oxadiazoles; Rabbits; Receptors, Nicotinic; Stereoisomerism; Thionucleotides