Activation of GABA B receptors in chick dorsal root ganglion (DRG) neurons inhibits the Ca v 2.2 calcium channel in both a voltage-dependent and voltage-independent manner. The voltage-independent inhibition requires activation of a tyrosine kinase that phosphorylates the ␣ 1 subunit of the channel and thereby recruits RGS12, a member of the "regulator of G protein signaling" (RGS) proteins. Here we report that RGS12 binds to the SNARE-binding or "synprint" region (amino acids 726 -985) in loop II-III of the calcium channel ␣1 subunit. A recombinant protein encompassing the Nterminal PTB domain of RGS12 binds to the synprint region in protein overlay and surface plasmon resonance binding assays; this interaction is dependent on tyrosine phosphorylation and yet is within a sequence that differs from the canonical NPXY motif targeted by other PTB domains. In electrophysiological experiments, microinjection of DRG neurons with synprint-derived peptides containing the tyrosine residue Tyr-804 altered the rate of desensitization of neurotransmitter-mediated inhibition of the Ca v 2.2 calcium channel, whereas peptides centered about a second tyrosine residue, Tyr-815, were without effect. RGS12 from a DRG neuron lysate was precipitated using synprint peptides containing phosphorylated Tyr-804. The high degree of conservation of Tyr-804 in the SNAREbinding region of Ca v 2.1 and Ca v 2.2 calcium channels suggests that this region, in addition to the binding of SNARE proteins, is also important for determining the time course of the modulation of calcium current via tyrosine phosphorylation.Multiple G protein-mediated signaling pathways are known to modulate Ca v 2.2 (N-type) calcium channels (1, 2) via direct G protein-ion channel interactions, activation of second messenger cascades, and activation of tyrosine kinases (3, 4). This modulation of voltage-dependent calcium channels is a transient phenomenon. Upon prolonged exposure to a neurotransmitter, neurons become unresponsive or desensitized. Despite the common requirement for the activation of a G proteincoupled receptor kinase (GRK3) for desensitization of the neurotransmitter-mediated inhibition of calcium current (5), G i -and G o -mediated pathways exhibit different rates of desensitization (6) that may result from selective effects of the G␣-directed GTPase-accelerating activity borne by "regulator of G protein signaling" (RGS) 1 proteins (7,8).In dorsal root ganglion (DRG) neurons, the activation of ␥-aminobutyric acid type B (GABA B ) receptors induces both voltage-dependent and voltage-independent inhibition of Ca v 2.2 channels (9). Voltage-independent inhibition requires the activation of a tyrosine kinase that phosphorylates the pore-forming ␣-subunit of the calcium channel (10). The tyrosine-phosphorylated form of the ␣-subunit becomes a target for the phosphotyrosine binding (PTB) domain of RGS12, a member of the RGS protein superfamily that specifically accelerates the rate of desensitization of this response (10).To better understand the molecular b...

Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAMankyrin B complex with the calcium channel mimics transmitterinduced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.Dunlap and Fischbach (1) have suggested that transmitter-mediated shortening of the duration of the action potential could be due to a decrease in calcium conductance or a decrease in the number of functional channels in the membrane. Because of the importance of such a mechanism for the regulation of synaptic transmission, much attention has been placed to the mechanisms of receptor-mediated modulation of voltage-gated calcium channels. Inhibition of Ca 2ϩ channels can be voltage-dependent and is mediated by direct interaction of G protein ␤␥ subunits with the ␣1 pore-forming subunit of the channel (2, 3). In addition, phosphorylation by kinases such as protein kinase C and tyrosine kinases has been shown to inhibit Ca 2ϩ channels (4). Subsequent work has established that G protein-dependent inhibition of calcium current is in part a result of a decrease in the open probability of the channel, reducing current density (5-7). The idea that changes in channel density could underlie calcium channel modulation has not been tested.Activity-and receptor-dependent trafficking of ionotropic receptors has been widely studied in the post-synaptic density (8, 9). Such studies have not been extended to proteins in the presynaptic active zones. In this study we have found that activation of G protein-coupled receptors induces destabilization and subsequent removal of calcium channels from the plasma membrane. Transmitter-induced trafficking of calcium channels is a consequence of disrupting the interaction of the channel with L1-CAM and ankyrin B and might represent a novel mechanism of presynaptic inhibition.

An emerging concept in signal transduction is the organization of neuronal receptors and channels into microdomains in which signaling proteins are brought together to regulate functional responses. With the multiplicity of potential protein-protein interactions arises the need for the regulation and timing of these interactions. We have identified N-type Ca 2؉ channelsignaling molecule complexes formed at different times upon activation of ␥-aminobutyric acid, type B, receptors. The first type of interaction involves pre-association of signaling proteins such as Src kinase with the Ca 2؉ channel, because it is rapidly activated by the receptors and regulates the magnitude of the inhibition of the Ca 2؉ channel. The second type of interaction involves signaling molecules that are recruited to the channel by receptor activation and control the rate of the channel response. Recruitment of members of the Ras pathway has two effects as follows: 1) modulation of the rate of onset of the ␥-aminobutyric acid-mediated inhibition of Ca 2؉ current, and 2) activation of MAP kinase. Our results suggest that the Ca 2؉ channel ␣ 1 subunit functions as a dynamic scaffold allowing assembly of intracellular signaling components that alter channel activity and route signals to the MAP kinase pathway.Voltage-dependent Ca 2ϩ channels are well known targets for inhibition by G protein-coupled receptors, and multiple pathways for inhibition have been described (1, 2). Inhibition of Ca 2ϩ channels can be voltage-dependent and is mediated by G protein ␤␥ subunits (1). In addition kinases, such as protein kinase C and tyrosine kinases, have been shown to inhibit Ca 2ϩ channels (2). As these multiple pathways converge in modulating Ca 2ϩ voltage-dependent channels, the question has arisen as to how these signals are integrated.Activation of tyrosine kinase by GABA B 1 receptors mediates voltage-independent inhibition of Ca v 2.2 (N-type) current in chick dorsal root ganglion (DRG) neurons (3), and the ␣ 1 subunit of the channel becomes tyrosine-phosphorylated (4). The tyrosine-phosphorylated channel binds to RGS12, a "regulator of G protein signaling" or RGS protein, that contains a phosphotyrosine binding domain (4). These findings raised the issue of whether tyrosine phosphorylation of the channel facilitates the recruitment of other signaling molecules that could then alter the functional characteristics of the channel and what role such interactions have in signal integration.We hypothesized that the Ca 2ϩ channel itself serves as an integrator by interacting with signaling molecules that regulate channel activity and by modulating downstream signals.To test this hypothesis, we have characterized interactions of the ␣ subunit of the Ca v 2.2 (N-type, ␣ 1B ) channel with signaling molecules, and we show how these interactions are regulated in an agonist and time-dependent manner and the consequences of such regulation on channel activity. Our results demonstrate that dynamic interactions at the level of the Ca 2ϩ channel can have shor...

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