Speed of Ca2+ channel modulation by neurotransmitters in rat sympathetic neurons

We have measured the onset and recovery speed of inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion neurons by somatostatin (SS), norepinephrine (NE), and oxotremorine-M (oxo-M, a muscarinic agonist), using the whole cell configuration of the patch-clamp method with 5 mM external Ca2+. With a local perfusion pipette system that changed the solution surrounding the cell within 50 ms, we applied agonists at various times before a brief depolarization from -80 mV that elicited I(Ca). At concentrations that produced maximal inhibition, the onset time constants for membrane-delimited inhibition by SS (0.5 microM), NE (10 microM), and oxo-M (20 microM) were 2.1, 0.7, and 1.0 s, respectively. The time constants for NE inhibition depended only weakly on the concentration, ranging from 1.2 to 0.4 s in the concentration range from 0.5 to 100 microM. Inhibition by oxo-M (20 microM) through a different G-protein pathway that uses a diffusible cytoplasmic messenger had a time constant near 9 s. The recovery rate constant from membrane-delimited inhibition was between 0.09 and 0.18 s(-1), significantly higher than the intrinsic GTPase rate of purified G protein Go, suggesting that Ca2+ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the rate of channel reinhibition after relief by strong depolarizing prepulses, which should reflect the kinetics of final steps in the inhibition process. In the presence of different concentrations of NE, reinhibition was four to seven times faster than the onset of inhibition, indicating that the slowest step of inhibition must precede the binding of G protein to the channel. We propose a kinetic model for the membrane-delimited NE inhibition of Ca2+ channels. It postulates two populations of receptors with different affinities for NE, a single population of G proteins, and a single population of Ca2+ channels. This model closely simulated the time courses of onset and recovery of inhibition and reinhibition, as well as the dose-response curve for inhibition of Ca2+ channels by NE.