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 somat ostatin (SS), norepinephrine (NE), and oxotremorine-M (oxo-M, a muscar inic agonist), using the whole cell configuration of the patch-clamp m ethod with 5 M external Ca2+. With a local perfusion pipette system th at changed the solution surrounding the cell within 50 ms, we applied agonists at various times before a brief depolarization from -80 mV th at elicited I-Ca. At concentrations that produced maximal inhibition, the onset time constants for membrane-delimited inhibition by SS (0.5 mu M), NE (10 mu M), and oxo-M (20 mu M) were 3.1, 0.7, and 1.0 s, res pectively. The time constants for NE inhibition depended only weakly o n the concentration, ranging rom 1.2 to 0.4 s in the concentration ran ge from 0.5 to 100 mu M. Inhibition by oxo-M (20 mu M) through a diffe rent C-protein pathway that uses a diffusible cytoplasmic messenger ha d a time constant near 9 s. The recovery rate constant from membrane-d elimited inhibition was between 0.09 and 0.18 s(-1), significantly hig her than the intrinsic GTPase rate of purified G protein G(0), suggest ing thar Ca2+ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the rate of channel rein hibition after relief by strong depolarizing prepulses, which should r eflect the kinetics of final steps in the inhibition process. Ln the p resence of different concentrations of NE, reinhibition was four to se ven times faster than the onset of inhibition, indicating that the slo west step of inhibition must precede the binding of G protein to the c hannel. We propose a kinetic model for the membrane-delimited NE inhib ition of Ca2+ channels. It postulates two populations of receptors wit h 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 .