G Protein-induced Trafficking of Voltage-dependent Calcium Channels

Abstract

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.

Arrestin Is Required for Agonist-induced Trafficking of Voltage-dependent Calcium Channels

“…S5). These results are not qualitatively different from those that we have previously obtained using rhodamine-conjugated -conotoxin GVIA (8).…”

“…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.…”

“…Whereas this internalization of calcium channels is a response to chronic exposure to an agonist, our observations on the spatiotemporal distribution of calcium channels have focused on early events after receptor activation (Ͻ1 min) (8). Agonist-induced desensitization of calcium current inhibition and return of the calcium channels to the surface occur in the time scale of 1 min (8,12).…”

“…The fast kinetics of internalization of calcium channels raises the question of whether components of the exocytic machinery exist in close proximity or pre-associated with the calcium channel. Whereas there is a high degree of co-localization between calcium channels and the endosomal markers Rab5 and clathrin heavy chain in cells exposed to agonist, there is a very low level of co-localization prior to receptor activation (8).…”

“…aging experiments in live embryonic chick DRG neurons have shown that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles (8). The fast kinetics of internalization of calcium channels raises the question of whether components of the exocytic machinery exist in close proximity or pre-associated with the calcium channel.…”