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.

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Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABA B receptor activation, receptors are recruited to the arrestin-channel complex and internalized. ␤-Arrestin 1 selectively binds to the SNAREbinding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.Inhibition of voltage-dependent calcium channels by seventransmembrane receptors (7TMR) 2 is one of the primary means of regulation of calcium-dependent physiological processes such as synaptic transmission, muscle contraction, and membrane excitability. In neurons, the Ca v 2.2 (N-type) channel is a prominent target for G protein-mediated modulation (1, 2). Inhibition of Ca v 2.2 channels can be voltage-dependent, and mediated by direct interactions with G protein ␤-␥ subunits (3, 4). In addition, kinases such as protein kinase C and tyrosine kinases have been shown to inhibit Ca v 2.2 channels in a voltage-independent manner (5, 6). Additional mechanisms may exist by which Ca 2ϩ influx is regulated. Dunlap and Fischbach (7) have suggested that transmitter-mediated shortening of the duration of the action potential could be due to a decrease in the number of voltage-dependent calcium channels at the membrane. Recently we have reported an additional mechanism by which 7TMRs can regulate neuronal calcium levels that involves a rapid internalization of voltage-dependent calcium channels into clathrin-coated vesicles upon receptor activation (8). Here we demonstrate that ␤-arrestin 1 is associated with Ca v 2.2 channels and that activation of 7TMRs results in the formation of an arrestin-receptor-channel complex. This interaction is required for internalization of calcium channels and plays a role in the modulation of calcium current. EXPERIMENTAL PROCEDURESMaterials-The following primary antibodies were used in these studies: rabbit anti-pan-␣ 1 (1:200,1.5 g/ml) (Alomone Labs, Jerusalem, Israel), anti-arrestin (1:500, BD Biosciences), and anti-GABAR1 (1:200, Chemicon). Anti-␤-arrestin 1 and anti-␤-arrestin 2 antibodies, and recombinant ␤-arrestin 1 and 2 (29) were kindly provided by the Lefkowitz laboratory. The following secondary antibodies were used in our studies: Oregon Green 488-conjugated goat anti-rabbit IgG (HϩL) (1:200, 10 g/ml), Cy3-conjugated goat anti-mouse IgG (HϩL) (1:200, 7.5 g/ml), and Cy5-goat anti-guinea pig IgG (HϩL) (1:200, 7.5 ...

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We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.