Biphasic, opposing modulation of cloned neuronal alpha1E Ca channels by distinct signaling pathways coupled to M2 muscarinic acetylcholine receptors.

Neuronal alpha1E subunits are thought to form R-type Ca channels. When expressed in human embryonic kidney cells with M2 muscarinic acetylcholine receptors, Ca channels encoded by rabbit alpha1E exhibit striking biphasic modulation. Receptor activation first produces rapid inhibition of current amplitude and activation rate. However, in the continued presence of agonist, alpha1E currents subsequently increase. Kinetic slowing persists during this secondary stimulation phase. After receptor deactivation, kinetic slowing is quickly relieved, and current amplitude over-recovers before returning toward control levels. These features indicate that inhibition and stimulation of alpha1E are separate processes, with stimulation superimposed on inhibition. Pertussis toxin eliminates inhibition without affecting stimulation, demonstrating that inhibition and stimulation involve distinct signaling pathways. Neither inhibition nor stimulation is altered by coexpression of Ca channel beta2a or beta3 subunits. Stimulation is abolished by staurosporine and reduced by intracellular 5'-adenylylimidodiphosphate, suggesting that phosphorylation is required. However, stimulation does not seem to involve cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, tyrosine kinases, or phosphoinositide 3-kinases. Stimulation does not require a Ca signal, because it is not specifically altered by varying intracellular Ca buffering or by substituting Ba as the charge carrier. In contrast to those formed by alpha1E, Ca channels formed by alpha1A or alpha1B display only inhibition and no stimulation during prolonged activation of M2 receptors. The dual modulation of alpha1E may confer unique physiological properties on native R-type Ca channels. As one possibility, R-type channels may continue to mediate Ca influx during steady inhibition of N-type and P/Q-type channels by muscarinic or other receptors.