Sonya M. Bierbower

Tmem100 Is a Regulator of TRPA1-TRPV1 Complex and Contributes to Persistent Pain.

TRPA1 and TRPV1 are crucial pain mediators, but how their interaction contributes to persistent pain is unknown. Here, we identify Tmem100 as a potentiating modulator of TRPA1-V1 complexes. Tmem100 is coexpressed and forms a complex with TRPA1 and TRPV1 in DRG neurons. Tmem100-deficient mice show a reduction in inflammatory mechanical hyperalgesia and TRPA1- but not TRPV1-mediated pain. Single-channel recording in a heterologous system reveals that Tmem100 selectively potentiates TRPA1 activity in a TRPV1-dependent manner. Mechanistically, Tmem100 weakens the association of TRPA1 and TRPV1, thereby releasing the inhibition of TRPA1 by TRPV1. A Tmem100 mutant, Tmem100-3Q, exerts the opposite effect; i.e., it enhances the association of TRPA1 and TRPV1 and strongly inhibits TRPA1. Strikingly, a cell-permeable peptide (CPP) containing the C-terminal sequence of Tmem100-3Q mimics its effect and inhibits persistent pain. Our study unveils a context-dependent modulation of the TRPA1-V1 complex, and Tmem100-3Q CPP is a promising pain therapy.

AKAP79/150 Signal Complexes in G-Protein Modulation of Neuronal Ion Channels

Voltage-gated M-type (KCNQ) K+ channels play critical roles in regulation of neuronal excitability. Previous work showed A-kinase-anchoring protein (AKAP)79/150-mediated protein kinase C (PKC) phosphorylation of M channels to be involved in M current (IM) suppression by muscarinic M1, but not bradykinin B2, receptors. In this study, we first explored whether purinergic and angiotensin suppression of IM in superior cervical ganglion (SCG) sympathetic neurons involves AKAP79/150. Transfection into rat SCG neurons of ΔA-AKAP79, which lacks the A domain necessary for PKC binding, or the absence of AKAP150 in AKAP150−/− mice, did not affect IM suppression by purinergic agonist or by bradykinin, but reduced IM suppression by muscarinic agonist and angiotensin II. Transfection of AKAP79, but not ΔA-AKAP79 or AKAP15, rescued suppression of IM by muscarinic receptors in AKAP150−/− neurons. We also tested association of AKAP79 with M1, B2, P2Y6, and AT1 receptors, and KCNQ2 and KCNQ3 channels, via Förster resonance energy transfer (FRET) on Chinese hamster ovary cells under total internal refection fluorescence microscopy, which revealed substantial FRET between AKAP79 and M1 or AT1 receptors, and with the channels, but only weak FRET with P2Y6 or B2 receptors. The involvement of AKAP79/150 in Gq/11-coupled muscarinic regulation of N- and L-type Ca2+ channels and by cAMP/protein kinase A was also studied. We found AKAP79/150 to not play a role in the former, but to be necessary for forskolin-induced upregulation of L-current. Thus, AKAP79/150 action correlates with the PIP2 (phosphatidylinositol 4,5-bisphosphate)-depletion mode of IM suppression, but does not generalize to Gq/11-mediated inhibition of N- or L-type Ca2+ channels.

β-Arrestin-2 Desensitizes the Transient Receptor Potential Vanilloid 1 (TRPV1) Channel

Background: The TRPV1 receptor is an ionotropic receptor implicated in a variety of pain and inflammatory disorders. Results: β-Arrestin-2 scaffolds phosphodiesterase PDE4D5 to TRPV1 to regulate receptor phosphorylation and activity. Conclusion: β-Arrestin-2 functions as a scaffold protein to mediate TRPV1 desensitization in multiple cell models. Significance: Our findings presented herein provide compelling support for the contribution of β-arrestins as scaffolding proteins in the regulation of ligand-gated ion channels. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel activated by multiple stimuli and is implicated in a variety of pain disorders. Dynamic sensitization of TRPV1 activity by A-kinase anchoring protein 150 demonstrates a critical role for scaffolding proteins in nociception, yet few studies have investigated scaffolding proteins capable of mediating receptor desensitization. In this study, we identify β-arrestin-2 as a scaffolding protein that regulates TRPV1 receptor activity. We report β-arrestin-2 association with TRPV1 in multiple cell models. Moreover, siRNA-mediated knockdown of β-arrestin-2 in primary cultures resulted in a significant increase in both initial and repeated responses to capsaicin. Electrophysiological analysis further revealed significant deficits in TRPV1 desensitization in primary cultures from β-arrestin-2 knock-out mice compared with wild type. In addition, we found that β-arrestin-2 scaffolding of phosphodiesterase PDE4D5 to the plasma membrane was required for TRPV1 desensitization. Importantly, inhibition of PDE4D5 activity reversed β-arrestin-2 desensitization of TRPV1. Together, these results identify a new endogenous scaffolding mechanism that regulates TRPV1 ligand binding and activation.

Activation of Mu Opioid Receptors Sensitizes Transient Receptor Potential Vanilloid Type 1 (TRPV1) via β-Arrestin-2-Mediated Cross-Talk

The transient receptor potential family V1 channel (TRPV1) is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C). Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs) on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

Augmentation of M-Type (KCNQ) Potassium Channels as a Novel Strategy to Reduce Stroke-Induced Brain Injury

Cerebral ischemic stroke is a worldwide cause of mortality/morbidity and thus an important focus of research to decrease the severity of brain injury. Therapeutic options for acute stroke are still limited. In neurons throughout the brain, “M-type” K+ currents, underlain by KCNQ subunits 2–5, play dominant roles in control over excitability, and are thus implicated in myriad neurological and psychiatric disorders. Although KCNQ channel openers, such as retigabine, have emerged as anti-epilepsy drugs, their effects on ischemic injury remain unknown. Here, we investigated the protective effects of M-channel openers on stroke-induced brain injury in mouse photothrombotic and middle cerebral artery occlusion (MCAo) models. Both photothrombosis and MCAo led to rapid, predictable, and consistently sized necrotic brain lesions, inflammatory responses, and behavioral deficits. Administration of three distinct M-channel openers at 0–6 h after ischemic injury significantly decreased brain infarct size and inflammation, and prevented neurological dysfunction, although they were more effective when administered 0–3 h poststroke. Thus, we show beneficial effects against stroke-induced brain injury and neuronal death through pharmacological regulation of ion channels that control neuronal excitability.

Pore Determinants of KCNQ3 K+ Current Expression

KCNQ3 homomeric channels yield very small macroscopic currents compared with other KCNQ channels or KCNQ2/3 heteromers. Two disparate regions of the channels--the C-terminus and the pore region--have been implicated in governing KCNQ current amplitudes. We previously showed that the C-terminus plays a secondary role compared with the pore region. Here, we confirm the critical role of the pore region in determining KCNQ3 currents. We find that mutations at the 312 position in the pore helix of KCNQ3 (I312E, I312K, and I312R) dramatically decreased KCNQ3 homomeric currents as well as heteromeric KCNQ2/3 currents. Evidence that these mutants were expressed in the heteromers includes shifted TEA sensitivity compared with KCNQ2 homomers. To test for differential membrane protein expression, we performed total internal reflection fluorescence imaging, which revealed only small differences that do not underlie the differences in macroscopic currents. To determine whether this mechanism generalizes to other KCNQ channels, we tested the effects of analogous mutations at the conserved I273 position in KCNQ2, with similar results. Finally, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels to investigate the putative structural mechanism mediating these results. The modeling suggests that the lack of current in I312E, I312K, and I312R KCNQ3 channels is due to pore helix-selectivity filter interactions that lock the selectivity filter in a nonconductive conformation.

Pore Helix-S6 Interactions Are Critical in Governing Current Amplitudes of KCNQ3 K+ Channels

Two mechanisms have been postulated to underlie KCNQ3 homomeric current amplitudes, which are small compared with those of KCNQ4 homomers and KCNQ2/Q3 heteromers. The first involves differential channel expression governed by the D-helix within the C-terminus. The second suggests similar channel surface expression but an intrinsically unstable KCNQ3 pore. Here, we find H2O2-enhanced oligomerization of KCNQ4 subunits, as reported by nondenaturing polyacrylamide gel electrophoresis, at C643 at the end of the D-helix, where KCNQ3 possesses a histidine. However, H2O2-mediated enhancement of KCNQ4 currents was identical in the C643A mutant, and KCNQ3 H646C produced homomeric or heteromeric (with KCNQ2) currents similar to those of wild-type KCNQ3, ruling out this divergent residue as underlying the small KCNQ3 amplitudes. In KcsA, F103 in S6 is critical for pore-mediated destabilization of the conductive pathway. We found that mutations at the analogous F344 in KCNQ3 dramatically decreased the KCNQ3 currents. Total internal reflection fluorescence imaging revealed only minor differential surface expression among the wild-type and mutant channels. Homology modeling suggests that the effects of the F344 mutants arise from the disruption of the interaction between F344 and A315 in the pore helix. These data support a secondary role of the C-terminus, compared with pore helix-S6 interactions, in governing KCNQ3 current amplitudes.

The Role of the Carboxyl Terminus Helix C-D Linker in Regulating KCNQ3 K+ Current Amplitudes by Controlling Channel Trafficking

In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies “M-type” currents, a slowly-activating voltage-gated K+ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum.

Förster Resonance Energy Transfer-Based Imaging at the Cell Surface of Live Cells

Understanding the molecular mechanisms of protein-protein interactions at the cell surface of living cells is fundamental to identifying the nature of cellular processes. Here, we discuss how fluorescence-based approaches have been successfully developed to visualize protein-protein interactions in living cells. Förster resonance energy transfer (FRET) is unique in generating fluorescence signals between proteins that are highly spatially sensitive. Furthermore, total internal reflectance fluorescence (TIRF) microscopy combined with FRET is a robust technique used to assay protein/protein interactions and the functionality of proteins assembled at the cell surface membrane.

Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulate KCNQ3 K+ Channels Via Multiple Sites Of Action

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the function of many ion channels, including M-type (KCNQ1-5, Kv7) K+ channels; however the molecular mechanisms involved in this regulation remain unclear. To identify the sites of action on KCNQ3 channels, we used as our baseline the A315T pore mutant (KCNQ3T) that increases channel currents without modifying the apparent affinity of PIP2 and performed extensive mutagenesis in regions that have been suggested to be involved in PIP2 interactions among the KCNQ family. Using the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe for apparent affinity of the channels, we found that PIP2 modulates KCNQ channel function through four different domains. 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix (S6Jx), 3) the S2-S3 linker and 4) the S4-S5 linker. We found that PIP2 interactions within these domains were not coupled to the voltage dependence of activation. Extensive homology modeling and docking simulations between the wild-type or mutant KCNQ3 channels and PIP2, correlated with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.