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: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:Potassium (K(+))-channel gating is choreographed by a complex interplay between external stimuli, K(+) concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K(+), pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K(+) and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K(+) ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K(+)-channel pore.
Project description:KCa3.1 (also known as SK4 or IK1) is a mammalian intermediate-conductance potassium channel that plays a critical role in the activation of T cells, B cells, and mast cells, effluxing potassium ions to maintain a negative membrane potential for influxing calcium ions. KCa3.1 shares primary sequence similarity with three other (low-conductance) potassium channels: KCa2.1, KCa2.2, and KCa2.3 (also known as SK1-3). These four homotetrameric channels bind calmodulin (CaM) in the cytoplasmic region, and calcium binding to CaM triggers channel activation. Unique to KCa3.1, activation also requires phosphorylation of a single histidine residue, His358, in the cytoplasmic region, which relieves copper-mediated inhibition of the channel. Near the cytoplasmic C-terminus of KCa3.1 (and KCa2.1-2.3), secondary-structure analysis predicts the presence of a coiled-coil/heptad repeat. Here, we report the crystal structure of the C-terminal coiled-coil region of KCa3.1, which forms a parallel four-helix bundle, consistent with the tetrameric nature of the channel. Interestingly, the four copies of a histidine residue, His389, in an 'a' position within the heptad repeat, are observed to bind a copper ion along the four-fold axis of the bundle. These results suggest that His358, the inhibitory histidine in KCa3.1, might coordinate a copper ion through a similar binding mode.
Project description:The sulphonylurea receptor (SUR) is a member of the ATP-binding cassette (ABC) family of membrane proteins. It functions as the regulatory subunit of the ATP-sensitive potassium (KATP) channel, which comprises SUR and Kir6.x proteins. Here, we review data demonstrating functional differences between the two nucleotide binding domains (NBDs) of SUR1. In addition, to explain the structural basis of these functional differences, we have constructed a molecular model of the NBD dimer of human SUR1. We discuss the experimental data in the context of this model, and show how the model can be used to design experiments aimed at elucidating the relationship between the structure and function of the KATP channel.