Phosducin and Phosducin-like Protein Attenuate G-Protein-Coupled Receptor-Mediated Inhibition of Voltage-Gated Calcium Channels in Rat Sympathetic Neurons

Abstract

Phosducin (PDC) has been shown in structural and biochemical experiments to bind the G␤␥ subunit of heterotrimeric G-proteins. A proposed function of PDC and phosducin-like protein (PDCL) is the sequestration of "free" G␤␥ from the plasma membrane, thereby terminating signaling by G␤␥. The functional impact of heterologously expressed PDC and PDCL on N-type calcium channel (Ca V 2.2) modulation was examined in sympathetic neurons, isolated from rat superior cervical ganglia, using whole-cell voltage clamp. Expression of PDC and PDCL attenuated voltage-dependent inhibition of N-type calcium channels, a G␤␥-dependent process, in a time-dependent fashion. Calcium current inhibition after short-term exposure to norepinephrine was minimally altered by PDC or PDCL expression. However, in the continued presence of norepinephrine, PDC or PDCL relieved calcium channel inhibition compared with control neurons. We observed similar results after activation of heterologously expressed metabotropic glutamate receptors with 100 M L-glutamate. Neurons expressing PDC or PDCL maintained suppression of inhibition after re-exposure to agonist. Unlike other G␤␥ sequestering proteins that abolish the short-term inhibition of Ca 2ϩ channels, PDC and PDCL require prolonged agonist exposure before effects on modulation are realized.G-protein-coupled receptors (GPCRs) are expressed throughout the nervous system and influence systems important for homeostasis. The canonical G-protein signaling pathways consists of a ligand binding to the receptor, exchange of GDP for GTP on the heterotrimeric G-protein ␣-subunit (G␣), separation of the G␤␥ dimer (G␤␥) from the G␣ subunit, and modulation of divergent downstream effector proteins by G␣-GTP and G␤␥. For example, free G␤␥ can associate with N-type voltage-gated Ca 2ϩ channels to reduce the probability of channel opening upon membrane depolarization. The strength and duration of this interaction will be influenced by competition with proteins possessing a high affinity for G␤␥, including G␣-GDP. Therefore, the duration of G-protein signaling is dependent upon the GTP hydrolysis rate because coalescence of the G␣␤␥ heterotrimer terminates signaling. Other proteins that bind G␤␥ with high affinity, such as the carboxyl terminus of GRK2 (ctGRK2) or phospholipase C-␤2, attenuate G␤␥ signaling (Ford et al., 1998). In this study, we examined how the expression of phosducins, a protein family known to interact with G␤␥, influenced GPCR-mediated Ntype Ca 2ϩ channel modulation in sympathetic neurons. The goals of the study were 1) to characterize the effects of PDC and PDCL expression within the context of protein-based optical sensor development and 2) to provide insight into possible physiological roles for PDC/PDCL modification of GPCR responses in neurons.Phosducin (PDC) is a 28-kDa soluble protein expressed primarily in the retina and pineal gland (Schulz, 2001;Sokolov et al., 2004). Retinal PDC has been hypothesized to adjust the gain of luminosity perception in the vertebrate eye (Thulin...

Phosducin Regulates the Expression of Transducin βγ Subunits in Rod Photoreceptors and Does Not Contribute to Phototransduction Adaptation

“…This idea was subsequently challenged by several reports indicating that most phosducin in the rod cell is located outside the outer segment, a photoreceptor's organelle where the visual signal transduction takes place (Lee et al, 1988;Gropp et al, 1997;Thulin et al, 1999;Nakano et al, 2001;Sokolov et al, 2004). Yet no direct evidence supporting or rejecting this putative mechanism has been reported so far and the hypothesis is still commonly discussed (e.g., Klenk et al, 2006;Partridge et al, 2006). On the other hand, phosducin was demonstrated to participate in another cellular function by assisting transducin βγ subunits in their light-driven translocation from rod outer segments (Sokolov et al, 2004), a process that takes place after prolonged exposure of rods to very bright light bleaching at least 4,000-6,000 rhodopsin molecules per rod per second (Sokolov et al, 2002;Lobanova et al, 2007;see Calvert et al, 2006 for a recent review).…”

The physiological roles of phosducin: from retinal function to stress-dependent hypertension

“…It has also been shown in cultured sympathetic neurons artificially expressing high levels of phosducin [141]. The concentration of phosducin in native sympathetic neurons is several orders of magnitude lower than in these tissues, which makes scavenging of bc subunits unlikely.…”

Utilizing GCaMP transgenic mice to monitor endogenous Gq/11-coupled receptors

“…In fact, GPCR activation has been observed with several imaging probe techniques including intramolecular and intermolecular Förster resonance energy transfer (FRET)-based genetically encoded indicators ( Lohse et al, 2012 ). However, an apparent constraint of this technique is that the introduction of dual fluorescent proteins (i.e., one acceptor and one donor), likely introduces steric hindrance and obstruction of essential protein–protein interactions necessary for energy transfer and the study of GPCRs in their endogenous states ( Partridge et al, 2006 ). By combining the fields of fluorescent microscopy, mouse genetics and pharmacology we can enhance our understanding of GPCRs in their native state.…”