No abstract

Gamma-aminobutyric acid (GABA)B receptors couple to Go to inhibit N-type calcium channels in embryonic chick dorsal root ganglion neurons. The voltage-independent inhibition, mediated by means of a tyrosine-kinase pathway, is transient and lasts up to 100 seconds. Inhibition of endogenous RGS12, a member of the family of regulators of G-protein signalling, selectively alters the time course of voltage-independent inhibition. The RGS12 protein, in addition to the RGS domain, contains PDZ and PTB domains. Fusion proteins containing the PTB domain of RGS12 alter the rate of termination of the GABA(B) signal, whereas the PDZ or RGS domains of RGS 12 have no observable effects. Using primary dorsal root ganglion neurons in culture, here we show an endogenous agonist-induced tyrosine-kinase-dependent complex of RGS12 and the calcium channel. These results indicate that RGS12 is a multifunctional protein capable of direct interactions through its PTB domain with the tyrosine-phosphorylated calcium channel. Recruitment of RGS proteins to G-protein effectors may represent an additional mechanism for signal termination in G-protein-coupled pathways.

The modulation of voltage-activated Ca2+ channels by neurotransmitters and peptides is very likely a primary means of regulating Ca(2+)-dependent physiological functions such as neurosecretion, muscle contraction, and membrane excitability. In neurons, N-type Ca2+ channels (defined as omega-conotoxin GVIA-sensitive) are one prominent target for transmitter-mediated inhibition. This inhibition is widely thought to result from a shift in the voltage independence of channel gating. Recently, however, voltage-independent inhibition has also been described for N channels. As embryonic chick dorsal root ganglion neurons express both of these biophysically distinct modulatory pathways, we have utilized these cells to test the hypothesis that the voltage-dependent and -independent actions of transmitters are mediated by separate biochemical pathways. We have confirmed this hypothesis by demonstrating that the two modulatory mechanisms activated by a single transmitter involve not only different classes of G protein but also different G protein subunits.

Neuronal Ca 2؉ channels are inhibited by a variety of transmitter receptors coupled to G o -type GTPbinding proteins. G o has been postulated to work via a direct interaction between an activated G protein subunit and the Ca 2؉ channel complex. Here we show that the inhibition of sensory neuron N-type Ca 2؉ channels produced by ␥-aminobutyric acid involves a novel, rapidly activating tyrosine kinase signaling pathway that is mediated by G␣ o and a src-like kinase. In contrast to other recently described G protein-coupled tyrosine kinase pathways, the G␣ o -mediated modulation requires neither protein kinase C nor intracellular Ca 2؉ . The results suggest that this pathway mediates rapid receptor-G protein signaling in the nervous system and support the existence of a previously unrecognized form of crosstalk between G protein and tyrosine kinase pathways.Receptor-mediated modulation of ion channels is an important means of regulating intercellular communication in the nervous system. Voltage-dependent Ca 2ϩ channels are effective targets for such modulation by virtue of their involvement in a host of cellular functions. Of the many calcium channel types now recognized, it has been the N-type (or class B) channels that have received the most attention in studies of receptor-mediated regulation. A large number of receptors and a variety of signaling pathways have been identified to target N channels (1, 2).High voltage-activated Ca 2ϩ currents evoked from cultured embryonic chicken sensory neurons are predominantly N-type (as they are blocked Ϸ90% by -conotoxin GVIA) and are inhibited by several neurotransmitters, including ␥-aminobutyric acid (GABA) and norepinephrine (NE). The inhibition is characterized by a slowing in the kinetics of N current activation (kinetic slowing) and an attenuation of the overall current amplitude without changes in current waveform (steady-state inhibition). Previous work has shown that these two components can be separated on the basis of a differential sensitivity to voltage (3).All inhibition by GABA is rapid and mediated through GABA B receptors (4) coupled to G proteins of the G o class (5). The speed of inhibition, coupled with the fact that it is independent of protein kinases A and C, cyclic nucleotides, intracellular Ca 2ϩ , and phosphatases 1 and 2A (6, 7), has led to the suggestion that GABA evokes inhibition through a direct effect of G protein subunits on the Ca 2ϩ channel (1, 2). Results reported below, however, suggest that steady-state inhibition is mediated indirectly through a novel tyrosine kinase pathway that is activated by G␣ o . MATERIALS AND METHODSEmbryonic chicken sensory neurons were grown in culture, and tight-seal whole-cell recording was performed after 1-3 days as in ref. 5. For intracellular application of kinase inhibitors, agents were diluted into intracellular recording solution and delivered to the cell via passive diffusion from the patch pipette. For extracellular application, agents were diluted into standard extracellular saline and ap...