Na_v1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Na_v1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Na_v1.9 (hNa_v1.9) was achieved by fusing GFP to the C-terminal of hNa_v1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological and pharmacological studies of hNa_v1.9. By using the hNa_v1.9 expression system, we investigated the electrophysiological properties of four mutations of hNa_v1.9 (K419N, A582T, A842P, and F1689L), whose electrophysiological functions have not been determined yet. The four mutations significantly caused positive shift of the steady-state fast inactivation and therefore increased hNa_v1.9 activity, consistent with the phenotype of painful peripheral neuropathy. Meanwhile, the effects of inflammatory mediators on hNa_v1.9 were also investigated. Impressively, histamine was found for the first time to enhance hNa_v1.9 activity, indicating its vital role in hNa_v1.9 modulating inflammatory pain. Taken together, our research provided a useful platform for hNa_v1.9 studies and new insight into mechanism of hNa_v1.9 linking to pain.

Background and Purpose: Blocking the voltage‐gated proton channel H_V1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few H_V1 channel antagonists have been reported. Here, we have identified a novel H_V1 channel antagonist from scorpion venom and have elucidated its action mechanism.Experimental Approach: H_V1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole‐cell patch clamp. Site‐directed mutagenesis was used to generate mutants. Toxins were recombinantly produced in Escherichia coli. AGAP/W38F‐H_V1 interaction was modelled by molecular dynamics simulations.Key Results: The scorpion toxin AGAP (anti‐tumour analgesic peptide) potently inhibited H_V1 currents. One AGAP mutant has reduced Na_V channel activity but intact H_V1 activity (AGAP/W38F). AGAP/W38F inhibited H_V1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited H_V1 currents with less pH dependence than Zn²⁺. Mutation analysis showed that the binding pockets of AGAP/W38F and Zn²⁺ in H_V1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in H_V1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn²⁺. Experimental data and MD simulations suggested that AGAP/W38F inhibited H_V1 channel using a Zn²⁺‐like long‐range conformational coupling mechanism.Conclusion and Implications: Our results suggest that the Zn²⁺ binding pocket in H_V1 channel might be a hotspot for modulators and valuable for designing H_V1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study H_V1 channel and a lead compound for drug development.

Antagonists — such as Ziconotide and Gabapentin — of the Ca _V 2.2 (N-type) calcium channels are used clinically as analgesics for chronic pain. However, their use is limited by narrow therapeutic windows, difficult dosing routes (Ziconotide), misuse, and overdoses (Gabapentin), as well as a litany of adverse effects. Expansion of novel pain therapeutics may emerge from mechanism-based interrogation of Ca _V 2.2. Here, we report the identification of C2230, an aryloxy-hydroxypropylamine, as a Ca _V 2.2 blocker. C2230 trapped and stabilized inactivated Ca _V 2.2 in a slow-recovering state and accelerated the open-state inactivation of the channel, conferring an advantageous use-dependent inhibition profile. C2230 inhibited Ca _V 2.2 during high-frequency stimulation, while sparing other voltage-gated ion channels. C2230 inhibited Ca _V 2.2 in dorsal root and trigeminal ganglia neurons from rats, marmosets, and humans in a G-protein-coupled-receptor–independent manner. Further, C2230 reduced evoked excitatory postsynaptic currents and excitatory neurotransmitter release in the spinal cord, leading to relief of neuropathic, orofacial, and osteoarthritic pain-like behaviors via 3 different routes of administration. C2230 also decreased fiber photometry-based calcium responses in the parabrachial nucleus, mitigated aversive behavioral responses to mechanical stimuli after neuropathic injury, and preserved protective pain responses, all without affecting motor or cardiovascular function. Finally, site-directed mutation analysis demonstrated that C2230 binds differently than other known Ca _V 2.2 blockers, making it a promising lead compound for analgesic development.

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000–4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na⁺, K⁺ and Ca²⁺ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na⁺ currents and T-type Ca²⁺ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD₅₀ was 84.24 μg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.

Pancreatic β cell failure is a hallmark of diabetes. However, the causes of β cell failure remain incomplete. Here, we report the identification of tetranectin (TN), an adipose tissue–enriched secretory molecule, as a negative regulator of insulin secretion in β cells in diabetes. TN expression is stimulated by high glucose in adipocytes via the p38 MAPK/TXNIP/thioredoxin/OCT4 signaling pathway, and elevated serum TN levels are associated with diabetes. TN treatment greatly exacerbates hyperglycemia in mice and suppresses glucose-stimulated insulin secretion in islets. Conversely, knockout of TN or neutralization of TN function notably improves insulin secretion and glucose tolerance in high-fat diet–fed mice. Mechanistically, TN binds with high selectivity to β cells and inhibits insulin secretion by blocking L-type Ca ²⁺ channels. Our study uncovers an adipocyte–β cell cross-talk that contributes to β cell dysfunction in diabetes and suggests that neutralization of TN levels may provide a new treatment strategy for type 2 diabetes.