Speed of Ca 2/ Channel Modulation by Neurotransmitters in Rat Sympathetic Neurons JIUYING ZHOU, MARK S. SHAPIRO, AND BERTIL HILLE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195-7290 Zhou, Jiuying, Mark S. Shapiro, and Bertil Hille. Speed of Ca 2/ ( Beech et al. 1992 ) . Muscarinic agonists, on the other hand, channel modulation by neurotransmitters in rat sympathetic neurons. also activate a pathway requiring a diffusible second messen- J. Neurophysiol. 77: 2040–2048, 1997. We have measured the onset ger. It uses a PTX-insensitive G protein, is voltage indepen- and recovery speed of inhibition of N-type Ca 2/ channels in adult rat dent, and is sensitive to calcium chelators in the recording superior cervical ganglion neurons by somatostatin ( SS), norepineph- pipette (the slow muscarinic pathway) (Beech et al. 1991; rine ( NE), and oxotremorine-M (oxo-M, a muscarinic agonist), using Bernheim et al. 1991). the whole cell configuration of the patch-clamp method with 5 mM To understand how modulation shapes dynamic responses external Ca 2/ . With a local perfusion pipette system that changed the of the nervous system, it is essential to know its kinetic solution surrounding the cell within 50 ms, we applied agonists at properties. Although previous work (Bernheim et al. 1991) various times before a brief depolarization from 080 mV that elicited I Ca . At concentrations that produced maximal inhibition, the onset has shown that inhibition through the membrane-delimited time constants for membrane-delimited inhibition by SS (0.5 mM), pathways is much faster than that through the second-mes- NE (10 mM), and oxo-M (20 mM) were 2.1, 0.7, and 1.0 s, respec- senger-requiring pathway, the speed of neurotransmitter in- tively. The time constants for NE inhibition depended only weakly hibition of Ca 2/ channels has not been extensively studied. on the concentration, ranging from 1.2 to 0.4 s in the concentration Here, we measured the onset and recovery kinetics of SS, range from 0.5 to 100 mM. Inhibition by oxo-M ( 20 mM) through a NE, and muscarinic inhibition of N-type Ca 2/ channels and different G-protein pathway that uses a diffusible cytoplasmic messen- its dependence on agonist concentration. We also measured ger had a time constant near 9 s. The recovery rate constant from the kinetics of the last steps in membrane-delimited channel membrane-delimited inhibition was between 0.09 and 0.18 s 01 , sig- inhibition by studying the rate of channel reinhibition after nificantly higher than the intrinsic GTPase rate of purified G protein a strong depolarizing pulse. We propose a kinetic model G o , suggesting that Ca 2/ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the describing membrane-delimited inhibition. rate of channel reinhibition after relief by strong depolarizing pre- pulses, which should reflect the kinetics of final steps in the inhibition METHODS process. In the presence of different concentrations of NE, reinhibition was four to seven times faster than the onset of inhibition, indicating Materials that the slowest step of inhibition must precede the binding of G Reagents were obtained as follows: papain (Worthington protein to the channel. We propose a kinetic model for the membrane- Biochemical), dispase (Boehringer Mannheim), somatostatin delimited NE inhibition of Ca 2/ channels. It postulates two popula- (Peninsula), oxotremorine-M (RBI), bis-( o-aminophenoxy)- tions of receptors with different affinities for NE, a single population N,N,N ,N -tetraacetic acid (BAPTA; Molecular Probes), leu- of G proteins, and a single population of Ca 2/ channels. This model peptin ( GIBCO ) , and ATP and GTP ( Pharmacia LKB Biotech- closely simulated the time courses of onset and recovery of inhibition nology). All other chemicals were from Sigma. and reinhibition, as well as the dose-response curve for inhibition of Ca 2/ channels by NE. Preparation of rat sympathetic neurons Neurons were dissociated acutely from the SCG of 4- to 6-wk- INTRODUCTION old Sprague-Dawley rats, using methods of Bernheim et al. ( 1991 ) , slightly modified by Shapiro and Hille (1993). Briefly, rats were Influx of extracellular Ca 2/ through voltage-gated Ca 2/ anesthetized with methoxyflurane and decapitated. Ganglia were channels is required for neurotransmitter release and also dissociated in modified Hank’s solution containing (in mM) 137 contributes to spike-frequency encoding. Therefore, depres- NaCl, 0.34 Na 2 HPO 4 r7H 2 O, 5.4 KCl, 0.44 KH 2 PO 4 , 5 glucose, sion of Ca 2/ channels by modulatory transmitters can play and 5 N-2-hydroxyethylpiperazine- N -2-ethanesulfonic acid important roles in regulation of neural signaling. In rat supe- (HEPES), pH 7.4 with NaOH. rior cervical ganglion ( SCG ) neurons, many neurotransmit- ters inhibit N-type Ca 2/ channels through at least five differ- Solutions ent intracellular pathways (for review, see Hille 1994). So- The external Ringer solution contained (in mM) 160 NaCl, 2.5 matostatin (SS), norepinephrine (NE), and muscarinic KCl, 5 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 8 glucose plus 500 nM agonists inhibit Ca 2/ channels through a widely studied sig- tetrodotoxin, pH adjusted to 7.4 with NaOH. The standard pipette naling pathway that uses pertussis toxin (PTX)-sensitive G solution contained (in mM) 175 CsCl, 5 MgCl 2 , 5 HEPES, 0,1 proteins and is membrane-delimited and voltage dependent BAPTA, 3 Na 2 ATP, 0.1 NaGTP, and 0.08 leupeptin, pH 7.4 with ( the standard pathway ) . NE also acts through another mem- CsOH. When measuring current inhibition by oxotremorine-M brane-delimited pathway that differs from this standard path- through the membrane-delimited pathway, 20 mM BAPTA was used in the pipette solution. When measuring the actions of NE, we way only in that the G protein involved is PTX insensitive 2040 0022-3077/97 $5.00 Copyright  1997 The American Physiological Society J782-6 / 9k0f$$ap40 08-27-97 15:03:36 neupal LP-Neurophys Downloaded from journals.physiology.org/journal/jn (003.081.235.037) on January 16, 2023.