okadaic-acid and 1-4-dihydropyridine

Dihydropyridine-sensitive, voltage-activated calcium channels respond to membrane depolarization with two distinct modes of activity: short bursts of very short openings (mode 1) or repetitive openings of much longer duration (mode 2). Here we show that both the dihydropyridine, BayK8644 (BayK), and the inhibitor of SerThr protein phosphatases, okadaic acid, have identical effects on the gating of the recombinant cardiac calcium channel, Ca(V)1.2 (alpha(1)C). Each produced identical mode 2 gating in cell-attached patches, and each prevented rundown of channel activity when the membrane patch was excised into ATP-free solutions. These effects required Ser or Thr at position 1142 in the domain III pore loop between transmembrane segments S5 and S6, where dihydropyridines bind to the channel. Mutation of Ser-1142 to Ala or Cys produced channels with very low activity that could not be modulated by either BayK or okadaic acid. A molecular model of Ca(V)1.2 indicates that Ser-1142 is unlikely to be phosphorylated, and thus we conclude that BayK binding stabilizes mode 2 gating allosterically by either protecting a phospho Ser/Thr on the alpha(1)C subunit or mimicking phosphorylation at that site.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Animals; Calcium Channel Agonists; Calcium Channels; Calcium Channels, L-Type; Cell Line; Cell Membrane; Cricetinae; Dihydropyridines; Electrophysiology; Enzyme Inhibitors; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Okadaic Acid; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Rabbits; Serine; Threonine; Time Factors; Transfection

During neuromuscular synapse development, the degradation rate of ACh receptors (AChRs) accumulated in the synaptic portion of the muscle membrane is drastically reduced under neural control, their half-life t1/2 increasing from 1 d to about 12 d. Recent evidence suggests that the metabolic stability of synaptic AChRs is mediated by the muscle activity induced by the nerve. We have now investigated the pathway linking muscle activity and metabolic stabilization of synaptic AChRs in organ cultured rat muscle. Soleus and diaphragm muscles were denervated for 14-40 d, a procedure leading to the destabilization of synaptic AChRs, and conditions required to restabilize synaptic AChRs in the denervated muscle were analyzed. The activity-dependent stabilization of synaptic AChRs in chronically denervated endplates required calcium entry through dihydropyridine-sensitive Ca2+ channels activated by high-frequency stimulation for approximately 6 hr and was specific for synaptic AChRs. As in vivo, extrasynaptic AChRs were not stabilized, and their t1/2 remained 1 d. The stabilization process was not dependent on de novo protein synthesis, and it could also be brought about by elevated cAMP levels. Furthermore, it required shorter stimulation periods in the presence of the phosphatase inhibitors okadaic acid and calyculin A, whereas blockade of protein kinases with high doses of staurosporine blocked the stabilization. Activity-dependent, dihydropyridine-sensitive as well as cAMP-dependent phosphorylation of myosin light chain was observed. These findings are consistent with the notion that muscle activity initiates AChR stabilization via the activation of calcium-dependent protein phosphorylation reactions.

Topics: Alkaloids; Animals; Bucladesine; Calcium; Calcium Channels; Cyclic AMP; Dibutyryl Cyclic GMP; Dihydropyridines; Electric Stimulation; Ethers, Cyclic; Kinetics; Male; Marine Toxins; Motor Endplate; Muscle Denervation; Muscles; Okadaic Acid; Organ Culture Techniques; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Potassium Chloride; Protein Kinase Inhibitors; Protein Kinases; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Staurosporine; Synapses; Tetrodotoxin; Time Factors