Orozco, I. | Search bioRxiv

bioRxiv2026

Biophysical Characterization of the human Nav1.9 sodium channel in trigeminal ganglia and dorsal root ganglia neurons

In human dorsal root ganglia (DRG), and trigeminal (TG) neurons, the various voltage-gated sodium channel (Nav) isoforms play critical roles in the firing of action potentials, which drive electrical impulses that encode somatosensations including, itch, and pain. The SCN11A gene encodes the tetrodotoxin (TTX)-resistant voltage-gated sodium channel Nav1.9, characterized by unique gating properties. Unlike other isoforms, the Nav1.9 channel activates and inactivates slowly and has a hyperpolarized voltage-dependence of activation and depolarized voltage-dependence of inactivation. This leads to a large window current that has been suggested to function as a regulator of the resting membrane potential of neurons. Mutations in Nav1.9 channels lead to congenital insensitivity to pain (gain-of-function) or familial episodic pain syndrome (loss-of-function) suggesting the channel is a critical mediator of pain. Despite its relevance in pain pathophysiology, most existing data relies on rodent models or heterologous expression systems, leaving the specific pharmacology and biophysical behavior of these channels in human primary neurons largely unknown. In this study, we pharmacologically isolated and characterized native Nav1.9 channel currents in human DRG and TG neurons to compare their biophysical profiles. Our findings reveal significant kinetic and voltage-dependent differences between the two populations. Specifically, Nav1.9 channels in TG neurons exhibit a right-shifted steady-state inactivation curve, a larger window current, and faster activation kinetics compared to those in DRG neurons. In addition, conditions that simulate inflammatory states in-vivo greatly potentiates the Nav1.9 currents consistent with similar observations in rodent models. By detailing these distinct biophysical properties, this research offers crucial insights into Nav1.9 channel function relevant for drug discovery efforts aimed at developing analgesics for both acute and chronic pain.

neuroscience↗

bioRxiv2021

Structures of TRPM5 channel elucidate mechanism of activation and inhibition

The Ca2+-activated TRPM5 channel plays an essential role in the perception of sweet, bitter, and umami stimuli in type II taste cells and in insulin secretion by pancreatic beta cells1-3. Interestingly, the voltage dependence of TRPM5 in taste bud cells depends on the intracellular Ca2+ concentration4, yet the mechanism remains elusive. Here we report cryo-electron microscopy structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state, at resolutions up to 2.3 [A]. We defined two novel ligand binding sites: a Ca2+ binding site (CaICD) in the intracellular domain (ICD), and an antagonist binding site in the transmembrane domain (TMD) for a drug (NDNA) that regulates insulin and GLP-1 release5. The CaICD site is unique to TRPM5 and has two roles: shifting the voltage dependence toward negative membrane potential, and promoting Ca2+ binding to the CaTMD site that is conserved throughout Ca2+-sensitive TRPM channels6. Replacing glutamate 337 in the CaICD site with an alanine not only abolished Ca2+ binding to CaICD but also reduced Ca2+ binding affinity to CaTMD, suggesting a cooperativity between the two sites. We have defined mechanisms underlying channel activation and inhibition. Conformational changes initialized from both Ca2+ sites, 70 [A] apart, are propagated to the ICD-TMD interface and cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the TMD in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents to treat metabolic disorders.

biophysics↗