ABSTRACT TRPV1 is a polymodal ion channel activated by vanilloids, noxious heat, and pro-inflammatory signals. A recent cryo-EM structure of human TRPV1 bound to SAF312, a potent, selective, noncompetitive antagonist, revealed a cholesterol molecule occupying the vanilloid-binding pocket, a site well established as the activation locus for vanilloid agonists. This observation led us to test whether cholesterol functionally inhibits capsaicin-dependent TRPV1 activation. Using HEK293 cells heterologously expressing TRPV1, we found that membrane cholesterol enrichment markedly suppressed capsaicin-evoked currents at low agonist concentrations, whereas responses to saturating capsaicin were unaffected. The functional interaction between cholesterol and capsaicin was further supported by site-directed mutagenesis targeting the conserved Gly563, a residue within the S4-S5 linker of the vanilloid-binding pocket. The G563S mutation reduced the sensitivity to capsaicin and caused slow and incomplete deactivation; nevertheless, elevated cholesterol further suppressed capsaicin-evoked activity. Together, these findings support a model in which cholesterol competes with capsaicin at the vanilloid-binding pocket to inhibit activation of the TRPV1 channel.

ABSTRACT The transient receptor potential vanilloid type 1 (TRPV1) channel, a member of the TRP ion channel family, plays a crucial role in both physiological and pathological processes. This review provides an overview of the structure, biological functions, and implications of TRPV1 in autoimmune diseases. The structural characteristics of TRPV1, including its transmembrane and intracellular domains, are examined to understand its activation and modulation. In addition to its well-known role as a thermosensor in nociceptive neurons, TRPV1 has been found to have functions in immune cells where it regulates lipid synthesis and inflammatory response. The investigation of TRPV1’s involvement in autoimmune conditions such as systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis highlights its potential as a therapeutic target. The search for selective agonists and antagonists for TRPV1 drugs is also discussed. A comprehensive understanding of TRPV1’s structure, function, and role in autoimmune diseases lays the foundation for future studies and the development of innovative therapies targeting this channel.

ABSTRACT TRPV4 is a polymodal Ca2+-permeable cation channel activated by diverse stimuli via various pathways and has been one of the difficult membrane proteins to comprehend, like other TRP channels. However, a broad range of functions and pathological conditions associated with these channels continues to fascinate researchers to study them. One of the major regulatory pathways of these channels is through protein phosphorylation catalyzed by various kinases (e.g. PKC, PKA, SGK1, and Src kinase) in a stimulus-specific manner. Several sites of protein phosphorylation have been identified in both N- and C-terminal tails located in the cytosolic region of the channel. One critical phosphorylation-mediated regulatory pathway involves the C-terminal phosphorylation of Ser-824 residue, which has been implicated in activation/sensitization of the channel and its functioning in cells. Due to the lack of structural evidence on the N- and C-terminal tails (largely intrinsically disordered), it remains a challenge to understand the molecular mechanisms involved in their regulation of the TRPV4 channel. However, recent studies have provided new insights into the potential mechanisms of phosphorylation regulation of the channel and helped unravel the complexity of TRPV4 regulation pathways. This review provides an updated summary of the regulatory role of post-translational regulation through phosphorylation, the kinases and residues involved in phosphorylation of the TRPV4 channel. Furthermore, we discuss the importance and potential mechanisms of the C-terminal domain, harboring the Ser-824 residue, in the regulation of channel activation and proper functioning.

ABSTRACT Intraocular pressure (IOP) is dynamically regulated by the contractility and viscoelasticity of the trabecular meshwork (TM). Two recent studies identified the polymodal cation channel TRPV4 as a central mechanosensor that integrates mechanical, biochemical, and circadian signals to set the IOP levels. Pharmacological TRPV4 inhibition, global Trpv4 knockout, and conditional deletion of Trpv4 attenuated pathological ocular hypertension induced by corticosteroids, TGFβ2, or angle occlusion, as well as physiological nocturnal IOP elevation. Conversely, the selective TRPV4 agonist GSK1016790A raised IOP when injected intracamerally but lowered it when applied topically, indicating compartment-specific action. TRPV4 activation induced actomyosin contractility and ECM deposition in cultured TM cells and increased outflow resistance in biomimetic 3D scaffolds and hydrogels, with the impact reversed by TRPV4 inhibition and gene deletion. TGFβ2 strongly upregulated transcription and functional expression of TRPV4, revealing a feed-forward fibrotic loop that may contribute to myofibroblast transdifferentiation of the stressed TM. Collectively, these findings established TRPV4 as an essential mediator of TM contractility, stiffness, and IOP homeostasis. Its expression in key pressure-regulating tissues (TM, Schlemm’s canal, ciliary body, and ciliary muscle) positions the channel as a convergence point for diverse glaucoma risk factors that regulate aqueous fluid production and drainage, and thus as a promising therapeutic target to lower IOP without global disruption of actin polymerization.

ABSTRACT Obesity is an established risk factor for atrial fibrillation (AF) and is associated with hypersecretion of the adipokine chemerin. Chemerin has been linked to the AF initiation and progression predominantly through Chemokine-like receptor 1(CMKLR1)-mediated signaling. This study aimed to elucidate how activation of the chemerin-CMKLR1 contributes to atrial potassium current dysregulation in obesity-related AF. Male C57BL/6J mice were divided into high-fat diet (HFD) and low-fat diet (LFD) group. Action potentials and potassium currents were recorded by whole-cell patch-clamp electrophysiology. HFD mice exhibited significantly increased susceptibility to AF. Atrial myocytes from HFD mice showed marked shortening of action potential duration, primarily due to an increase in peak repolarizing potassium current (I k,peak). The rise in I K,peak density was attributed to concurrent remodeling of its components, the transient outward potassium current (I to) and the ultrarapid delayed rectifier potassium current (I KUr). I to density increased from 30.13 ± 0.76 pA/pF to 35.42 ± 0.70 pA/pF at +70 mV, accompanied by a leftward shift of steady-state activation, a rightward shift of steady-state inactivation, faster recovery from inactivation, and upregulated Kv4.3 and KChIP2 expression. I KUr density increased from 23.95 ± 1.95 pA/pF to 30.24 ± 0.97 pA/pF at +70 mV, consistent with elevated Kv1.5 expression. These electrophysiological changes were paralleled by upregulated protein abundance of chemerin and its receptor CMKLR1 in atrial myocytes, suggesting activation of the chemerin-CMKLR1 in obese mice. Obesity-associated activation of the chemerin-CMKLR1 promotes pathological potassium current remodeling, shortens atrial APD, and contributes to obesity-related AF.

ABSTRACT The modulator pocket is a cryptic site discovered in the TREK1 (K2P2.1) K2P channel. This pocket, located close to the selectivity filter, accommodates agonists that enhance the channel’s activity. Since its discovery, equivalent sites in other K2P channels have been shown to bind various ligands, both endogenous and exogenous. In this review, we attempt to elucidate how the modulator pocket contributes to K2P channel activation. To this end, we first describe the gating mechanisms reported in the literature and rationalize their modes of action. We then highlight previous experimental and computational evidence for agonists that bind to the modulator pocket, together with mutations at this site that affect gating. Finally, we elaborate how the activation signal arising from the modulator pocket is transduced to the gates in K2P channels. In doing so, we outline a potential common modulator pocket architecture across K2P channels: a largely amphipathic structure – consistent with the expected properties of a pocket exposed at the interface between a hydrophobic membrane and the aqueous solvent – but still with some important channel-sequence-variations. This architecture and its key differences can be leveraged for the design of new selective and potent modulators.

ABSTRACT Neuronal function requires fine-tuned and coordinated activity of several ion channels and transporters. One member of this ensemble is the KCC2 potassium-chloride cotransporter. Because KCC2 expression is required for GABA-dependent inhibitory synaptic transmission, mutations in the gene encoding KCC2 (SLC12A5) have been linked to several diseases that also arise from defects in GABA signaling, including epilepsy, schizophrenia, and autism spectrum disorders. Although characterization of the corresponding mutant proteins is ongoing, KCC2 mutants may reside at the cell surface but lack function, they may remain trapped intracellularly and are thus unable to function at the cell surface, or they may be readily degraded. In this article, we summarize these data and emphasize the importance of protein degradation and protease activity during KCC2 quality control, i.e. the pathway that ensures only properly folded and mature KCC2 can traffic to and function at the cell surface. We also highlight how proteolysis regulates the amount of active KCC2 at the cell surface, i.e. KCC2 quantity control. Finally, because previously unidentified KCC2 mutants are continuously being discovered, we discuss the use of predictive pathogenicity algorithms to provide researchers with information on potential disease outcomes.

ABSTRACT Voltage-gated CaV2.2 channels underlie the N-type current, and they regulate calcium entry at many presynaptic nerve endings to control transmitter release. A role for CaV2.2 channels has been well established in the transmission of sensory signals including noxious information using pharmacological and global gene knockout mouse models. However, investigation of the cell-specific actions of CaV2.2 channels has been difficult due to the lack of gene-dependent knockout mouse models and particularly in dissecting behavioral responses that depend on CaV2.2 channel activity. Here, we show the importance of CaV2.2 channels in Trpv1-lineage neurons in behavioral responses to sensory stimuli using Cre-dependent inactivation of the Cacna1b gene. Our work shows the cell-type specificity of CaV2.2 channels in mediating rapidly developing heat hypersensitivity and the utility of Cre-dependent inactivation of Cacna1b to discern cell-specific CaV2.2 channel functions.

ABSTRACT Kv10.1 is a voltage-gated K+ channel whose structure–function relationships remain incompletely understood, and whose ectopic expression is linked to tumorigenesis. We have recently shown that the antiarrhythmic drug amiodarone inhibits both the K+ current and the characteristic Cole–Moore shift of Kv10.1. Here, we examined whether the amiodarone derivative KB130015 similarly modulates Kv10.1 function. Low micromolar concentrations of KB130015 markedly accelerated current activation across all tested holding potentials and fully abolished the Cole–Moore shift. The t1⁄2 reduction induced by KB130015 was voltage independent. KB130015 also slowed channel deactivation to a similar extent at all voltages and shifted the G–V relationship toward more negative potentials without altering its slope. Despite these pronounced gating effects, current amplitude increased only slightly and showed minimal dependence on KB130015 concentration. Notably, KB130015 enhanced the inhibitory effect of amiodarone on K+ current. These results identify KB130015 as a potent modulator of Kv10.1 gating that also potentiates amiodarone-mediated inhibition.

ABSTRACT In the European Union, urinary incontinence (UI) affects 45% of adults during their lifetime, representing a major clinical and socio-economic burden. Failure of urethral smooth muscle (USM) to contract normally (hypo or hypercontractility) contributes to UI symptoms such as urine leakage during bladder filling or inability to urinate due to obstruction. Adequate UI treatments are lacking, partially due to a lack in understanding of cellular mechanisms underlying USM contraction. USM contractions rely on Ca2+ signaling in urethral smooth muscle cells (USMC), resulting from Ca2+ release from internal stores and Ca2+ influx from extracellular sources, such as voltage-gated L-type Ca2+ channels or store-operated Ca2+ entry (SOCE) channels. L-type Ca2+ channel inhibitors have inconsistent effects on urethral contractions across species, including humans, and thus solely targeting this pathway may be insufficient to modulate USM contractility. Recent animal experiments suggest SOCE mediated by Orai-STIM proteins is a critical determinant of Ca2+ signaling in USMC, maintaining regenerative Ca2+ release from internal stores, and thus may be a targetable pathway for influencing USM contractility. In this review, we highlight evidence suggesting SOCE as critical for Ca2+ signaling in USMC from multiple species and propose possible mechanisms for how this occurs at the cellular level.