Project description:Beta-adrenergic receptor stimulation increases heart rate and shortens ventricular action-potential duration, the latter effect due in part to a cAMP-dependent increase in the slow outward potassium current (I(Ks)). Mutations in either KCNQ1 or KCNE1, the I(Ks) subunits, are associated with variants (LQT-1 and LQT-5) of the congenital long QT syndrome. We now show that cAMP-mediated functional regulation of KCNQ1/KCNE1 channels, a consequence of cAMP-dependent protein kinase A phosphorylation of the KCNQ1 N terminus, requires coexpression of KCNQ1 with KCNE1, its auxiliary subunit. Further, at least two KCNE1 mutations linked to LQT-5 (D76N and W87R) cause functional disruption of cAMP-mediated KCNQ1/KCNE1-channel regulation despite the response of the substrate protein (KCNQ1) to protein kinase A phosphorylation. Transduction of protein phosphorylation into physiologically necessary channel function represents a previously uncharacterized role for the KCNE1 auxiliary subunit, which can be disrupted in LQT-5.
Project description:G protein-coupled receptor 37-like 1 (GPR37L1) is an orphan GPCR, and its function remains largely unknown. Here we report that Gpr37l1 and GPR37L1 are among the most highly expressed GPCR transcripts in mouse and human dorsal root ganglia (DRGs) and are selectively expressed in satellite glial cells (SGCs). Peripheral neuropathy following PTX-induced pain resulted in a downregulation of GPR37L1 plasma membrane expression in DRGs. Transgenic mice with Gpr37l1 deficiency exhibited impaired resolution of neuropathic pain symptoms following PTX-induced pain, whereas overexpression of Gpr37l1 in mouse DRGs reversed pain. GPR37L1 regulates the surface expression and function of these potassium channels. Thus, GPR37L1 in SGCs offers a new target for neuropathy protection and pain control.
Project description:Cyclic nucleotide-binding (CNB) domains regulate the activity of channels, kinases, exchange factors, and transcription factors. These proteins are highly variable in their ligand selectivity; some are highly selective for either cAMP or cGMP, whereas others are not. Several molecular determinants of ligand selectivity in CNB domains have been defined, but these do not provide a complete view of the selectivity mechanism. We performed a thorough analysis of the ligand-binding properties of mutants of the CNB domain from the MlotiK1 potassium channel. In particular, we defined which residues specifically favor cGMP or cAMP. Inversion of ligand selectivity, from favoring cAMP to favoring cGMP, was only achieved through a combination of three mutations in the ligand-binding pocket. We determined the x-ray structure of the triple mutant bound to cGMP and performed molecular dynamics simulations and a biochemical analysis of the effect of the mutations. We concluded that the increase in cGMP affinity and selectivity does not result simply from direct interactions between the nucleotide base and the amino acids introduced in the ligand-binding pocket residues. Rather, tighter cGMP binding over cAMP results from the polar chemical character of the mutations, from greater accessibility of water molecules to the ligand in the bound state, and from an increase in the structural flexibility of the mutated binding pocket.
Project description:Human ether-à-go-go-related gene (hERG, KCNH2) voltage-activated potassium channels are critical for cardiac excitability. hERG channels have characteristic slow closing (deactivation), which is auto-regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). hERG channels are not activated by the binding of extrinsic cyclic nucleotide ligands, but rather bind an "intrinsic ligand" that is composed of residues 860-862 within the CNBHD and mimics a cyclic nucleotide. The intrinsic ligand is located at the PAS-CNBHD interface, but its mechanism of action in hERG is not well understood. Here we use whole-cell patch-clamp electrophysiology and FRET spectroscopy to examine how the intrinsic ligand regulates gating. To carry out this work, we coexpress PAS (a PAS domain fused to cyan fluorescent protein) in trans with hERG "core" channels (channels with a deletion of the PAS domain fused to citrine fluorescent protein). The PAS domain in trans with hERG core channels has slow (regulated) deactivation, like that of WT hERG channels, as well as robust FRET, which indicates there is a direct functional and structural interaction of the PAS domain with the channel core. In contrast, PAS in trans with hERG F860A core channels has intermediate deactivation and intermediate FRET, indicating perturbation of the PAS domain interaction with the CNBHD. Furthermore, PAS in trans with hERG L862A core channels, or PAS in trans with hERG F860G,L862G core channels, has fast (nonregulated) deactivation and no measurable FRET, indicating abolition of the PAS and CNBHD interaction. These results indicate that the intrinsic ligand is necessary for the functional and structural interaction between the PAS domain and the CNBHD, which regulates the characteristic slow deactivation gating in hERG channels.
Project description:KCNH voltage-gated potassium channels play critical roles in regulating cellular functions. The channel is composed of four subunits, each of which contains six transmembrane helices forming the central pore. The cytoplasmic parts of the subunits present a Per-Arnt-Sim (PAS) domain at the N-terminus and a cyclic nucleotide-binding homology domain at the C-terminus. PAS domains are conserved from prokaryotes to eukaryotes and are involved in sensing signals and cellular responses. To better understand the functional roles of PAS domains in KCNH channels, the structure of this domain from the human ether-à-go-go channel (hEAG channel) was determined. By comparing it with the structures of the Homo sapiens EAG-related gene (hERG) channel and the Drosophila EAG-like K(+) (dELK) channel and analyzing the structural features of the hEAG channel, it was identified that a hydrophobic patch on the β-sheet may mediate interaction between the PAS domain and other regions of the channel to regulate its functions.
Project description:BackgroundIn radiotherapy, 2D orthogonally projected kV images are used for patient alignment when 3D-on-board imaging (OBI) is unavailable. However, tumor visibility is constrained due to the projection of patient's anatomy onto a 2D plane, potentially leading to substantial setup errors. In treatment room with 3D-OBI such as cone beam CT (CBCT), the field of view (FOV) of CBCT is limited with unnecessarily high imaging dose. A solution to this dilemma is to reconstruct 3D CT from kV images obtained at the treatment position.MethodsWe propose a dual-models framework built with hierarchical ViT blocks. Unlike a proof-of-concept approach, our framework considers kV images acquired by 2D imaging devices in the treatment room as the solo input and can synthesize accurate, full-size 3D CT within milliseconds.ResultsWe demonstrate the feasibility of the proposed approach on 10 patients with head and neck (H&N) cancer using image quality (MAE: < 45HU), dosimetric accuracy (Gamma passing rate ((2%/2 mm/10%): > 97%) and patient position uncertainty (shift error: < 0.4 mm).ConclusionsThe proposed framework can generate accurate 3D CT faithfully mirroring patient position effectively, thus substantially improving patient setup accuracy, keeping imaging dose minimal, and maintaining treatment veracity.
Project description:Background and purposeOur initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity.Experimental approachFollowing the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans.Key resultsVSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity.Conclusions and implicationsWe identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity.
Project description:Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1-S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an 'open' conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.
Project description:G protein-coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gβγ subunits). Therefore, G protein modifiers are well positioned to shape GPCR pharmacology. A few members of the potassium channel tetramerization domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gβγ. However, comprehensive details on the KCTD interaction with Gβγ remain unresolved. Here, we report that nearly all the 25 KCTD proteins interact with Gβγ. In this study, we screened Gβγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell bioluminescence resonance energy transfer-based assay, we find that roughly half of KCTD proteins interact with Gβγ in an agonist-induced fashion, whereas all KCTD proteins except two were found to interact through coimmunoprecipitation. We observed that the interaction was dependent on an amino acid hot spot in the C terminus of KCTD2, KCTD5, and KCTD17. While KCTD2 and KCTD5 require both the Bric-à-brac, Tramtrack, Broad complex domain and C-terminal regions for Gβγ interaction, we uncovered that the KCTD17 C terminus is sufficient for Gβγ interaction. Finally, we demonstrated the functional consequence of the KCTD-Gβγ interaction by examining sensitization of the adenylyl cyclase-cAMP pathway in live cells. We found that Gβγ-mediated sensitization of adenylyl cyclase 5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gβγ to shape GPCR signal transmission.
Project description:Ever since they were first observed in Purkinje fibers of the heart, funny channels have had close connections to potassium channels. Indeed, funny channels were initially thought to produce a potassium current in the heart called I K2. However, funny channels are completely unlike potassium channels in ways that make their contributions to the physiology of cells unique. An important difference is the greater ability for sodium to permeate funny channels. Although it does not flow through the funny channel as easily as does potassium, sodium does permeate well enough to allow for depolarization of cells following a strong hyperpolarization. This is critical for the function of funny channels in places like the heart and brain. Computational analyses using recent structures of the funny channels have provided a possible mechanism for their unusual permeation properties.