Gut enterochromaffin cells drive visceral pain and anxiety.

Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain1. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved2. Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men1. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.

Crypt and Villus Enterochromaffin Cells are Distinct Stress Sensors in the Gut.

The crypt-villus structure of the small intestine serves as an essential protective barrier, with its integrity monitored by the gut's sensory system. Enterochromaffin (EC) cells, which are rare sensory epithelial cells that release serotonin (5-HT), surveil the mucosal environment and signal both within and outside the gut. However, it remains unclear whether EC cells in intestinal crypts and villi respond to different stimuli and elicit distinct responses. In this study, we introduce a new reporter mouse model to observe the release and propagation of serotonin in live intestines. Using this system, we show that crypt EC cells exhibit two modes of serotonin release: transient receptor potential A1 (TRPA1)-dependent tonic serotonin release that controls basal ionic secretion, and irritant-evoked serotonin release that activates gut sensory neurons. Furthermore, we find that a thick protective mucus layer prevents TRPA1 receptors on crypt EC cells from responding to luminal irritants such as reactive electrophiles; if this mucus layer is compromised, then crypt EC cells become susceptible to activation by luminal irritants. On the other hand, villus EC cells detect oxidative stress through TRPM2 channels and co-release serotonin and ATP to activate nearby gut sensory fibers. Our work highlights the physiological importance of intestinal architecture and differential TRP channel expression in sensing noxious stimuli that elicit nausea and/or pain sensations in the gut.

Evidence that toxin resistance in poison birds and frogs is not rooted in sodium channel mutations and may rely on "toxin sponge" proteins.

Many poisonous organisms carry small-molecule toxins that alter voltage-gated sodium channel (NaV) function. Among these, batrachotoxin (BTX) from Pitohui poison birds and Phyllobates poison frogs stands out because of its lethality and unusual effects on NaV function. How these toxin-bearing organisms avoid autointoxication remains poorly understood. In poison frogs, a NaV DIVS6 pore-forming helix N-to-T mutation has been proposed as the BTX resistance mechanism. Here, we show that this variant is absent from Pitohui and poison frog NaVs, incurs a strong cost compromising channel function, and fails to produce BTX-resistant channels in poison frog NaVs. We also show that captivity-raised poison frogs are resistant to two NaV-directed toxins, BTX and saxitoxin (STX), even though they bear NaVs sensitive to both. Moreover, we demonstrate that the amphibian STX "toxin sponge" protein saxiphilin is able to protect and rescue NaVs from block by STX. Taken together, our data contradict the hypothesis that BTX autoresistance is rooted in the DIVS6 N→T mutation, challenge the idea that ion channel mutations are a primary driver of toxin resistance, and suggest the possibility that toxin sequestration mechanisms may be key for protecting poisonous species from the action of small-molecule toxins.

A Selectivity Filter Gate Controls Voltage-Gated Calcium Channel Calcium-Dependent Inactivation.

Calcium-dependent inactivation (CDI) is a fundamental autoregulatory mechanism in CaV1 and CaV2 voltage-gated calcium channels. Although CDI initiates with the cytoplasmic calcium sensor, how this event causes CDI has been elusive. Here, we show that a conserved selectivity filter (SF) domain II (DII) aspartate is essential for CDI. Mutation of this residue essentially eliminates CDI and leaves key channel biophysical characteristics untouched. DII mutants regain CDI by placing an aspartate at the analogous SF site in DIII or DIV, but not DI, indicating that CaV SF asymmetry is key to CDI. Together, our data establish that the CaV SF is the CDI endpoint. Discovery of this SF CDI gate recasts the CaV inactivation paradigm, placing it squarely in the framework of voltage-gated ion channel (VGIC) superfamily members in which SF-based gating is important. This commonality suggests that SF inactivation is an ancient process arising from the shared VGIC pore architecture.

A Calmodulin C-Lobe Ca2+-Dependent Switch Governs Kv7 Channel Function.

Kv7 (KCNQ) voltage-gated potassium channels control excitability in the brain, heart, and ear. Calmodulin (CaM) is crucial for Kv7 function, but how this calcium sensor affects activity has remained unclear. Here, we present X-ray crystallographic analysis of CaM:Kv7.4 and CaM:Kv7.5 AB domain complexes that reveal an Apo/CaM clamp conformation and calcium binding preferences. These structures, combined with small-angle X-ray scattering, biochemical, and functional studies, establish a regulatory mechanism for Kv7 CaM modulation based on a common architecture in which a CaM C-lobe calcium-dependent switch releases a shared Apo/CaM clamp conformation. This C-lobe switch inhibits voltage-dependent activation of Kv7.4 and Kv7.5 but facilitates Kv7.1, demonstrating that mechanism is shared by Kv7 isoforms despite the different directions of CaM modulation. Our findings provide a unified framework for understanding how CaM controls different Kv7 isoforms and highlight the role of membrane proximal domains for controlling voltage-gated channel function. VIDEO ABSTRACT.

Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic ß-subunit (CaVß). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:CaVß interactions and reduce the entropic penalty associated with AID binding to CaVß. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVß interaction that modulate CaV function in an CaVß isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based CaV modulator design.