Robyn Flynn

Regulation of calcium channels by RGK proteins

The RGK family of proteins, small GTPases of the Ras superfamily, are known to regulate calcium currents. Iit is commonly thought that this is due to an interaction with the Cavβ subunit, however, the mechanism of this inhibition is unclear. There have been conflicting reports of whether RGK proteins can affect channel trafficking or whether they reduce calcium currents by interacting with channels at the membrane. In the last year, several studies have emerged which explore the intricacies of RGK protein interaction with the channel itself and the importance of the Cavβ subunit for this interaction, in addition to providing some tantalizing suggestions for the mechanism by which RGK proteins reduce or eliminate calcium currents. In this review, we present an overview of these recent advances and suggest a model that may synthesize these latest works.

TRPV1 sensitization mediates postinflammatory visceral pain following acute colitis

Quiescent phases of inflammatory bowel disease (IBD) are often accompanied by chronic abdominal pain. Although the transient receptor potential vanilloid 1 (TRPV1) ion channel has been postulated as an important mediator of visceral hypersensitivity, its functional role in postinflammatory pain remains elusive. This study aimed at establishing the role of TRPV1 in the peripheral sensitization underlying chronic visceral pain in the context of colitis. Wild-type and TRPV1-deficient mice were separated into three groups (control, acute colitis, and recovery), and experimental colitis was induced by oral administration of dextran sulfate sodium (DSS). Recovery mice showed increased chemically and mechanically evoked visceral hypersensitivity 5 wk post-DSS discontinuation, at which point inflammation had completely resolved. Significant changes in nonevoked pain-related behaviors could also be observed in these animals, indicative of persistent discomfort. These behavioral changes correlated with elevated colonic levels of substance P (SP) and TRPV1 in recovery mice, thus leading to the hypothesis that SP could sensitize TRPV1 function. In vitro experiments revealed that prolonged exposure to SP could indeed sensitize capsaicin-evoked currents in both cultured neurons and TRPV1-transfected human embryonic kidney (HEK) cells, a mechanism that involved TRPV1 ubiquitination and subsequent accumulation at the plasma membrane. Importantly, although TRPV1-deficient animals experienced similar disease severity and pain as wild-type mice in the acute phase of colitis, TRPV1 deletion prevented the development of postinflammatory visceral hypersensitivity and pain-associated behaviors. Collectively, our results suggest that chronic exposure of colon-innervating primary afferents to SP could sensitize TRPV1 and thus participate in the establishment of persistent abdominal pain following acute inflammation.

Activity-driven mobilization of post-synaptic proteins

Synapses established during central nervous system development can be modified through synapse elimination and formation. These processes are, in part, activity dependent and require regulated trafficking of post‐synaptic components. Here, we investigate the activity‐driven remodeling of cultured rat hippocampal neurons at 14 days in vitro, focusing on the post‐synaptic proteins PSD‐95, Shank, neuroligin (NL)1 and actin. Using live imaging and photoconductive stimulation, we found that high‐frequency activity altered the trajectory, but not velocity, of PSD‐95‐GFP and Shank‐YFP clusters, whereas it reduced the speed and increased the number of NL1 clusters. Actin‐CFP reorganized into puncta following activity and ∼50% of new puncta colocalized with NL1 clusters. Actin reorganization was enhanced by the overexpression of NL1 and decreased by the expression of an NL1 mutant, NL1‐R473C. These results demonstrate activity‐dependent changes that may result in the formation of new post‐synaptic sites and suggest that NL1 modulates actin reorganization. The results also suggest that a common mechanism underlies both the developmental and activity‐dependent remodeling of excitatory synapses.

Molecular determinants of Rem2 regulation of N-type calcium channels

Rem2 belongs to the RGK family of small GTPases whose members are known to interact with the voltage gated calcium channel beta subunit, and to inhibit or abolish calcium currents. To identify the underlying functional domains of Rem2, we created several N- or C-terminally truncated Rem2 proteins and examined their abilities to interact with the Ca(v) beta subunit and to regulate the activities of Ca(v)2.2 N-type calcium channels. Confocal imaging of Rem2 in tsA-201 cells revealed that it contains a membrane-targeting signal in its C-terminus, consistent with previous studies. Co-precipitation assays showed that Ca(v) beta(3) interaction depends on Rem2 residues 1-123. Only Rem2 proteins that targeted the cell membrane as well as bound the beta subunit were able to reduce whole cell calcium currents.

Targeting the transient receptor potential vanilloid type 1 (TRPV1) assembly domain attenuates inflammation-induced hypersensitivity.

Background: Assembly of TRPV1 subunits forms functional channels that transduce noxious stimuli. Results: We identified a region of the TRPV1 C terminus that mediates subunit assembly and used a disrupting peptide to block association. Conclusion: A short C-terminal motif enables subunit association. Disrupting assembly of TRPV1 subunits attenuates inflammatory hyperalgesia. Significance: TRPV1 subunit association could be targeted for pain relief. The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief.

Activity-dependent subcellular cotrafficking of the small GTPase Rem2 and Ca2+/CaM-dependent protein kinase IIα.

Background Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners. Results We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca2+ influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca2+/CaM-dependent protein kinase IIα (CaMKII) a in Ca2+/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells. Conclusions Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity.

A macromolecular trafficking complex composed of β2-adrenergic receptors, A-Kinase Anchoring Proteins and L-type calcium channels

Abstract Sympathetic modulation of cardiac L-type calcium channels is an important mechanism for regulating heart rate and cardiac contractility. At the molecular level, activation of β-adrenergic receptors (βAR) increases calcium influx into cardiac myocytes by activating protein kinase A (PKA), leading to subsequent phosphorylation of L-type calcium channels. In the case of the β2AR, this process is facilitated by the presence of A-Kinase Anchoring Proteins (AKAPs) that serve as scaffolding proteins for the L-type calcium channel and the β2AR complex. Our work has shown that, in addition to facilitating PKA phosphorylation of the channel, AKAPs also promote an increase in the Cav1.2 channel surface expression. Here we review the molecular mechanisms of β2AR/AKAP/L-type channel interactions and trafficking.

The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel

Background Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund’s Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. Results We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund’s Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Conclusions Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function and points to the role of this stress protein in pain associated with neurodegenerative and/or metabolic disorders, including aging.

TRPV1 regulates opioid analgesia during inflammation

Acute inflammation in humans or mice enhances the analgesic properties of opioids. However, the inflammatory transducers that prime opioid receptor signaling in nociceptors are unknown. We found that TRPV1−/− mice are insensitive to peripheral opioid analgesia in an inflammatory pain model. We report that TRPV1 channel activation drives a MAPK signaling pathway accompanied by the shuttling of β-arrestin2 to the nucleus. This shuttling in turn prevents: β-arrestin2-receptor recruitment, subsequent internalization of agonist-bound mu opioid receptor (MOR), and suppression of DAMGO-induced inhibition of N-type calcium current observed upon desensitization. Consequently, inflammation-induced activation of TRPV1 preserves opioid analgesic potency in a mouse model of opioid receptor desensitization. Overall, our work reveals a TRPV1-mediated signaling mechanism, involving β-arrestin2 nuclear translocation, that underlies the peripheral opioid control of inflammatory pain. Our data single out TRPV1 channels as modulators of opioid analgesia.