In silico assessment of interaction of sea anemone toxin APETx2 and acid sensing ion channel 3

Acid sensing ion channels (ASICs) are proton-gated cation channels that are expressed throughout the nervous system and have been implicated in mediating sensory perception of noxious stimuli. Amongst the six ASIC isoforms, ASIC1a, 1b, 2a and 3 form proton-gated homomers, which differ in their activation and inactivation kinetics, expression profiles and pharmacological modulation; protons do not gate ASIC2b and ASIC4. As with many other ion channels, structure-function studies of ASICs have been greatly aided by the discovery of some toxins that act in isoform-specific ways. ASIC3 is predominantly expressed by sensory neurons of the peripheral nervous system where it acts to detect acid as a noxious stimulus and thus plays an important role in nociception. ASIC3 is the only ASIC subunit that is inhibited by the sea anemone (Anthopleura elegantissima)-derived toxin APETx2. However, the molecular mechanism by which APETx2 interacts with ASIC3 remains largely unknown. In this study, we made a homology model of ASIC3 and used extensive protein-protein docking to predict for the first time, the probable sites of APETx2 interaction on ASIC3. Additionally, using computational alanine scanning, we also suggest the 'hot-spots' that are likely to be critical for ASIC3-APETx2 interaction.T.R. is a recipient of a fellowship and additional support funding (RG120370) from the Royal Society

Expression of acid-sensing ion channels and selection of reference genes in mouse and naked mole rat.

Acid-sensing ion channels (ASICs) are a family of ion channels comprised of six subunits encoded by four genes and they are expressed throughout the peripheral and central nervous systems. ASICs have been implicated in a wide range of physiological and pathophysiological processes: pain, breathing, synaptic plasticity and excitotoxicity. Unlike mice and humans, naked mole-rats do not perceive acid as a noxious stimulus, even though their sensory neurons express functional ASICs, likely an adaptation to living in a hypercapnic subterranean environment. Previous studies of ASIC expression in the mammalian nervous system have often not examined all subunits, or have failed to adequately quantify expression between tissues; to date there has been no attempt to determine ASIC expression in the central nervous system of the naked mole-rat. Here we perform a geNorm study to identify reliable housekeeping genes in both mouse and naked mole-rat and then use quantitative real-time PCR to estimate the relative amounts of ASIC transcripts in different tissues of both species. We identify RPL13A (ribosomal protein L13A) and CANX (calnexin), and β-ACTIN and EIF4A (eukaryotic initiation factor 4a) as being the most stably expressed housekeeping genes in mouse and naked mole-rat, respectively. In both species, ASIC3 was most highly expressed in dorsal root ganglia (DRG), and ASIC1a, ASIC2b and ASIC3 were more highly expressed across all brain regions compared to the other subunits. We also show that ASIC4, a proton-insensitive subunit of relatively unknown function, was highly expressed in all mouse tissues apart from DRG and hippocampus, but was by contrast the lowliest expressed ASIC in all naked mole-rat tissues

The naked mole-rat as an animal model in biomedical research: current perspectives

The naked mole-rat (NMR) is a subterranean rodent that has gained significant attention from the biomedical research community in recent years as molecular mechanisms underlying its unusual biology start to be unraveled. With very low external mortality, NMRs have an unusually long lifespan while showing no signs of aging, such as neurodegeneration or cancer. Furthermore, living underground in large colonies (100 to 300 animals), results in comparatively high carbon dioxide and low oxygen levels, from which NMRs have evolved extreme resistance to both hypoxia and hypercapnia. In this paper we have summarized the latest developments in NMR research and its impact on biomedical research, with the aim of providing a sound background that will inform and inspire further investigations

Absence of histamine-induced itch in the African naked mole-rat and "rescue" by Substance P

Recent research has proposed a pathway in which sensory neurons expressing the capsaicin activated ion channel TRPV1 are required for histamine-induced itch and subsequent scratching behavior. We examined histamine-induced itch in the African naked mole-rat (Heterocephalus glaber) and found that although naked mole-rats display innate scratching behavior, histamine was unable to evoke increased scratching as is observed in most mouse strains. Using calcium imaging, we examined the histamine sensitivity of naked mole-rat dorsal root ganglia (DRG) neurons and identified a population of small diameter neurons activated by histamine, the majority of which are also capsaicinsensitive. This suggested that naked mole-rat sensory neurons are activated by histamine, but that spinal dorsal horn processing of sensory information is not the same as in other rodents. We have previously shown that naked mole-rats naturally lack substance P (SP) in cutaneous C-fibers, but that the neurokinin-1 receptor is expressed in the superficial spinal cord. This led us to investigate if SP deficiency plays a role in the lack of histamine-induced scratching in this species. After intrathecal administration of SP into the spinal cord we observed robust scratching behavior in response to histamine injection. Our data therefore support a model in which TRPV1-expressing sensory neurons are important for histamine-induced itch. In addition, we demonstrate a requirement for active, SP-induced post-synaptic drive to enable histamine sensitive afferents to drive itch-related behavior in the naked mole-rat. These results illustrate that it is altered dorsal horn connectivity of nociceptors that underlies the lack of itch and pain-related behavior in the naked mole-rat.This work was supported by the Alexander von Humboldt Foundation (EStJS) and NSF grant 0744979 (TJP)

Subunit-specific inhibition of acid sensing ion channels by stomatin-like protein 1

There are five mammalian stomatin-domain genes, all of which encode peripheral membrane proteins that can modulate ion channel function. Here we examined the ability of stomatin-like protein 1 (STOML1) to modulate the proton-sensitive members of the acid-sensing ion channel (ASIC) family. STOML1 profoundly inhibits ASIC1a, but has no effect on the splice variant ASIC1b. The inactivation time constant of ASIC3 is also accelerated by STOML1. We examined STOML1 null mutant mice with a β-galactosidase-neomycin cassette gene-trap reporter driven from the STOML1 gene locus, which indicated that STOML1 is expressed in at least 50% of dorsal root ganglion (DRG) neurones. Patch clamp recordings from mouse DRG neurones identified a trend for larger proton-gated currents in neurones lacking STOML1, which was due to a contribution of effects upon both transient and sustained currents, at different pH, a finding consistent with an endogenous inhibitory function for STOML1.This project was supported by grants of the German Research Council (SFB449/B18 and SFB958/A09 to G.R.L.)

An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors

Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.This work was supported by grants from the DFG to I.I.-T. and G.R.L. within the collaborative research centre (SFB 665) and from the Alexander von Humboldt Foundation to E.St.J.S

Microfluidic SAXS study of lamellar and multilamellar vesicle phases of linear sodium alkylbenzenesulfonate surfactant with intrinsic isomeric distribution

The structure and flow behaviour of a concentrated aqueous solution (45 w.t. %) of the ubiquitous linear sodium alkylbenzene sulfonate (NaLAS) surfactant is investigated by microfluidic small-angle X-ray scatterong (SAXS) at 70 ⁰C. NaLAS is an intrinsically complex mixture of over 20 surfactant molecules, presenting coexisting micellar (L1) and lamellar (Lα) phases. Novel microfluidic devices were fabricated to ensure pressure and thermal resistance, ability to handle viscous fluids, and low SAXS background. Polarized light optical microscopy showed that the NaLAS solution exhibits wall slip in microchannels, with velocity profiles approaching plug flow. Microfluidic SAXS demonstrated the structural spatial heterogeneity of the system with a characteristic lengthscale of 50 nL. Using a statistical flow-SAXS analysis we identified the micellar phase and multiple coexisting lamellar phases with a continuous distribution of d spacings between 37.5 Å - 39.5 Å. Additionally, we showed that the orientation of NaLAS lamellar phases is strongly affected by a single microfluidic constriction. The bilayers align parallel to the velocity field upon entering a constriction and perpendicular to it upon exiting. On the other hand, multi-lamellar vesicle phases are not affected under the same flow conditions. Our results demonstrate that, despite the compositional complexity inherent to NaLAS, microfluidic SAXS can rigorously elucidate its structure and flow response

Peripheral KV7 channels regulate visceral sensory function in mouse and human colon

$\textbf{Background}$ Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The K$_V$7 family (K$_V$7.1-K$_V$7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral K$_V$7 channels to visceral nociception. $\textbf{Results}$ Immunohistochemical staining of mouse colon revealed labelling of K$_V$7 subtypes (K$_V$7.3 and K$_V$7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the K$_V$7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B$_2$ bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0-80 mmHg) in a concentration-dependent manner, whereas the K$_V$7 blocker XE991 potentiated such responses. In human bowel tissues, K$_V$7.3 and K$_V$7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. $\textbf{Conclusions}$ We show that K$_V$7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted K$_V$7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies.This work was supported by an EFIC-Grunenthal grant awarded to Madusha Peiris, CAG/CIHR/CCFR Fellowship to David E Reed, Rosetrees Postdoctoral Grant (A1296) to James RF Hockley/Ewan St. John Smith, Medical Research Council Grant (G0900907) to David C Bulmer and a Wellcome Trust University Award to L Ashley Blackshaw

Atomic force microscopy imaging reveals the formation of ASIC/ENaC cross-clade ion channels

ASIC and ENaC are co-expressed in various cell types, and there is evidence for a close association between them. Here, we used atomic force microscopy (AFM) to determine whether ASIC1a and ENaC subunits are able to form cross-clade hybrid ion channels. ASIC1a and ENaC could be co-isolated from detergent extracts of tsA 201 cells co-expressing the two subunits. Isolated proteins were incubated with antibodies against ENaC and Fab fragments against ASIC1a. AFM imaging revealed proteins that were decorated by both an antibody and a Fab fragment with an angle of ∼120° between them, indicating the formation of ASIC1a/ENaC heterotrimers.J.M.E. and P.J. are supported by Kidney Research U.K. A.P.S. was a member of the University of Cambridge MB/PhD Programme, and was supported by the Jean Shanks Foundation and the James Baird Fund. A research stay of S.H. at Cambridge was supported by a PhD fellowship from the Bayerische Forschungsstiftung

ASICs and mammalian mechanoreceptor function

It is well established that some members of the Deg/ENaC super family of amiloride sensitive ion channels can participate directly in the transduction of mechanical stimuli by sensory neurons in invertebrates. A large body of work has also implicated the acid sensing ion channels family (ASIC1-4) as participants in regulating mechanoreceptor sensitivity in vertebrates. In this review we provide an overview of the physiological and genetic evidence for involvement of ASICs in mechanosensory function. On balance, the available evidence favors the idea that these channels have an important regulatory role in mechanosensory function. It is striking how diverse the consequences of Asic gene deletion are on mechanosensory function with both gain and loss of function effects being observed depending on sensory neuron type. We conclude that other, as yet unknown, molecular partners of ASIC proteins may be decisive in determining their precise physiological role in mechanosensory neurons. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'.Work in the Lewin lab was supported by a senior ERC award (Grant no 294678) and grants from the Deutsche Forschungsgemeinshaft (SFB665 and SFB958). Y-A. B. S. was funded by a fellowship from the Deutsche akademsische Austausch Dienst (DAAD). E. St. J. S. is funded by an Early Career Research Grant from the International Association for the Study of Pain. L-N. S is funded by the BBSRC Doctoral Training Program and the David James studentship