Voltage-dependent calcium channels are well-known targets for inhibition by G protein-coupled receptors (GPCRs), and multiple forms of inhibition have been described. Inhibition of Ca²⁺ channels can be voltage-dependent, and is mediated by direct interaction of G protein beta-gamma subunits with the alpha 1 pore-forming subunit of the channel (Ikeda, 1996; Herlitze et al., 1996). In addition phosphorylation by kinases, such as protein kinase C and tyrosine kinases have been shown to inhibit Ca²⁺ channels (Dolphin, 2003). The molecular entities and mechanisms that control the rate-limiting steps underlying the onset and the termination of receptor-mediated channel modulation, are not fully understood. My laboratory studies the integration of the multiple signaling pathways that converge to modulate calcium channel activity and how they could ultimately alter the timing of signaling in the nervous system. The overall goal of my laboratory is to identify the components of signaling complexes regulating calcium channel function, and to understand how interactions of these components with the calcium channel determine the time course of transmitter-mediated inhibition of calcium influx in neurons.

Molecular determinants of desensitization of transmitter-mediated modulation of calcium channels

Transmitter-induced inhibition of Cav2.2 channels is a transient phenomenon. We have shown that the onset and rate of desensitization of transmitter-mediated modulation of calcium channels are mediated by different molecular loci. The onset of desensitization requires the activation of an endogenous G protein-coupled receptor kinase (GRK) similar to the beta adrenergic receptor kinase type 2 (also called GRK3). The rate of desensitization is determined by the activity of a family of GTPase accelerating proteins, regulators of G protein signaling (RGS) proteins. These results suggest that different molecular complexes control the onset and the rate of desensitization.

We have described a new mechanism of desensitization at the level of the Ca²⁺ channel. Tyrosine kinase-mediated phosphorylation of G protein effectors, such as calcium channels, is involved in the termination of G protein-mediated signaling by facilitating interactions of the effector with other proteins which might regulate channel activity. Tyrosine kinases can act both as "on" and "off" molecular switches within the G protein pathway. During onset of the response, Src kinase phosphorylates the alpha subunit of the calcium channel. The tyrosine-phosphorylated calcium channel becomes a target for binding of the phosphotyrosine-binding (PTB) domain of RGS12. Recent experiments from our laboratory have shown that RGS12 binds to the SNARE-binding region of the calcium channel; on-going experiments are to determine whether RGS12 competes with the SNARE proteins for binding to the channel. Our results suggest that RGS12 is a multifunctional protein capable of direct interactions with Ca²⁺channels. Interactions between RGS proteins and effectors, independent of regulation of GTP hydrolysis by G protein alpha-subunits, represents a hitherto unrecognized mechanism for signal termination in G protein coupled pathways.

Agonist-induced internalization of calcium channels

Recently we have found that activation of GPCRs can induce endocytosis of voltage-dependent calcium channels. Time-lapse experiments in which calcium channels from chick DRG neurons were labeled with rhodamine-conjugated ω-conotoxin GVIA have shown that, within two seconds of transmitter application, the channels move away from the surface of the plasma membrane. We have found that calcium channels interact with ankyrin B and the cell adhesion molecule L1-CAM under basal conditions and that this interaction is abrogated by GPCR activation.

Voltage-dependent calcium channels are pre-associated with beta-arrestin 1, a protein known to play a role in receptor trafficking. 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.