Rat brain cannabinoid receptor (CB-1) was stably transfected into the murine tumor line AtT-20 to study its coupling to inwardly rectifying potassium currents (Kir) and high voltage-activated calcium currents (ICa). In cells expressing CB-1 ("A-2" cells), cannabinoid agonist potently and stereospecifically activated Kir via a pertussis toxin-sensitive G protein. ICa in A-2 cells was sensitive to dihydropyridines and omega CTX MVIIC, less so to omega CgTX GVIA and insensitive to omega Aga IVa. In CB-1 expressing cells, cannabinoid agonist inhibited only the omega CTX MVIIC-sensitive component of ICa. Inhibition of Q-type ICa was voltage dependent and PTX sensitive, thus similar in character to the well-studied modulation of N-type ICa. An endogenous cannabinoid, anandamide, activated Kir and inhibited ICa as efficaciously as potent cannabinoid agonist. Immunocytochemical studies with antibodies specific for class A, B, C, D, and E voltage-dependent calcium channel alpha 1 subunits revealed that AtT-20 cells express each of these major classes of alpha 1 subunit.
Ca2+/calmodulin-dependent protein kinase H contains two subunits, a (Mr 50,000) and 13 (Mr 60,000/ 58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. In the present study, we have studied the mechanism of this autophosphorylation reaction and its effect on the activity of the enzyme. Both subunits are autophosphorylated through an intramolecular mechanism. Using synapsin
Membrane vesicles enriched in both ryanodine receptor and dihydropyridine receptor were obtained from rabbit skeletal muscle and solubilized with 3- [(3-cholamidopropyl) (14,17,18).Three different mechanisms have been proposed for excitation-contraction coupling in skeletal muscle: Ca2 -induced Ca2+ release, inositol 1,4,5-trisphosphate-induced Ca2W release, and direct physical coupling (for review see refs. 19 and 20). Current evidence favors a model in which a voltagedriven conformational change ofthe al subunit ofthe DHP-R activates Ca2+ release by the RyR through direct physical interaction (11,21,22
Ca2+ influx through skeletal muscle Ca2+ channels and the force of contraction are increased in response to beta-adrenergic stimulation and high-frequency electrical stimulation. These effects are thought to be mediated by cAMP-dependent phosphorylation of the skeletal muscle Ca2+ channel. Modulation of the cloned skeletal muscle Ca2+ channel by cAMP-dependent phosphorylation and by depolarizing prepulses was reconstituted by transient expression in tsA-201 cells and compared to modulation of the native skeletal muscle Ca2+ channel as expressed in mouse 129CB3 skeletal muscle cells. The heterologously expressed Ca2+ channel consisting of alpha1, alpha2delta, and beta subunits gave currents that were similar in time course, current density, and dihydropyridine sensitivity to the native Ca2+ channel. cAMP-dependent protein kinase (PKA) stimulation by Sp-5,6-DCl-cBIMPS (cBIMPS) increased currents through both native and expressed channels two- to fourfold. Tail currents after depolarizations to potentials between -20 and +80 mV increased in amplitude and decayed more slowly as either the duration or potential of the depolarization was increased. The time- and voltage-dependent slowing of channel deactivation required the activity of PKA, because it was enhanced by cBIMPS and reduced or eliminated by the peptide PKA inhibitor PKI (5-24) amide. This voltage-dependent modulation of the cloned skeletal muscle Ca2+ channel by PKA also required anchoring of PKA by A-Kinase Anchoring Proteins because it was blocked by peptide Ht 31, which disrupts such anchoring. The results show that the skeletal muscle Ca2+ channel expressed in heterologous cells is modulated by PKA at rest and during depolarization and that this modulation requires anchored protein kinase, as it does in native skeletal muscle cells.
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