An unorthodox mechanism underlying voltage sensitivity of TRPV1 ion channel

While the capsaicin receptor TRPV1 channel is a polymodal nociceptor for heat, capsaicin, and proton, the channel’s responses to each of these stimuli are profoundly regulated by membrane potential, damping or even prohibiting its response at negative voltages and amplifying its response at positive voltages. Though voltage sensitivity plays an important role is shaping pain responses, how voltage regulates TRPV1 activation remains unknown. Here we showed that the voltage sensitivity of TRPV1 does not originate from the S4 segment like classic voltage-gated ion channels; instead, outer pore acidic residues directly partake in voltage-sensitive activation, with their negative charges collectively constituting the observed gating charges. Voltage-sensitive outer pore movement is titratable by extracellular pH and is allosterically coupled to channel activation, likely by influencing the upper gate in the ion selectivity filter. Elucidating this unorthodox voltage-gating process provides a mechanistic foundation for understanding polymodal gating and opens the door to novel approaches regulating channel activity for pain managements.

Gαq and Phospholipase Cβ signaling regulate nociceptor sensitivity in Drosophila melanogaster larvae

Drosophila melanogaster larvae detect noxious thermal and mechanical stimuli in their environment using polymodal nociceptor neurons whose dendrites tile the larval body wall. Activation of these nociceptors by potentially tissue-damaging stimuli elicits a stereotyped escape locomotion response. The cellular and molecular mechanisms that regulate nociceptor function are increasingly well understood, but gaps remain in our knowledge of the broad mechanisms that control nociceptor sensitivity. In this study, we use cell-specific knockdown and overexpression to show that nociceptor sensitivity to noxious thermal and mechanical stimuli is correlated with levels of Gαq and phospholipase Cβ signaling. Genetic manipulation of these signaling mechanisms does not result in changes in nociceptor morphology, suggesting that changes in nociceptor function do not arise from changes in nociceptor development, but instead from changes in nociceptor activity. These results demonstrate roles for Gαq and phospholipase Cβ signaling in facilitating the basal sensitivity of the larval nociceptors to noxious thermal and mechanical stimuli and suggest future studies to investigate how these signaling mechanisms may participate in neuromodulation of sensory function.

Proton block of proton-activated TRPV1 current

The TRPV1 cation channel is a polymodal nociceptor that is activated by heat and ligands such as capsaicin and is highly sensitive to changes in extracellular pH. In the body core, where temperature is usually stable and capsaicin is normally absent, H+ released in response to ischemia, tissue injury, or inflammation is the best-known endogenous TRPV1 agonist, activating the channel to mediate pain and vasodilation. Paradoxically, removal of H+ elicits a transient increase in TRPV1 current that is much larger than the initial H+-activated current. We found that this prominent OFF response is caused by rapid recovery from H+ inhibition of the excitatory current carried by H+-activated TRPV1 channels. H+ inhibited current by interfering with ion permeation. The degree of inhibition is voltage and permeant ion dependent, and it can be affected but not eliminated by mutations to acidic residues within or near the ion selectivity filter. The opposing H+-mediated gating and permeation effects produce complex current responses under different cellular conditions that are expected to greatly affect the response of nociceptive neurons and other TRPV1-expressing cells.

Proton Sensitivity Ca2+ Permeability and Molecular Basis of Acid-Sensing Ion Channels Expressed in Glabrous and Hairy Skin Afferents

We contrasted the physiology and peripheral targets of subclassified nociceptive and nonnociceptive afferents that express acid-sensing ion channel (ASIC)–like currents. The threshold for current activation was similar in eight distinct cell subclasses regardless of functional modality (pH 6.8). When potency was determined from concentration–response curves, nonnociceptors exhibited currents with significantly greater potency than that of all but one class of nociceptors (pH50 = 6.54 and 6.75 vs. 6.20–6.34). In nonnociceptive cells, acid transduction was also confined to a very narrow range (0.1–0.3 vs. 0.8–1.4 pH units for nociceptors). Simultaneous whole cell recording and ratiometric imaging of three peptidergic nociceptive classes were consistent with the expression of Ca2+-permeable ASICs. Sensitivity to psalmotoxin and flurbiprofen indicated the presence of Ca2+-permeable ASIC1a. Immunocytochemistry on these subclassified populations revealed a differential distribution of five ASIC proteins consistent with Ca2+ permeability and differential kinetics of proton-gated currents (type 5: ASIC1a, 1b, 2a, 2b, 3; type 8a: ASIC1a, 1b, 3; type 8b: ASIC1a, 1b, 2a, 2b, 3). Using DiI tracing, we found that nociceptive classes had discrete peripheral targets. ASIC-expressing types 8a and 9 projected to hairy skin, but only types 8a and 13 projected to glabrous skin. Non-ASIC–expressing types 2 and 4 were present only in hairy skin. We conclude that ASIC-expressing nociceptors differ from ASIC-expressing nonnociceptors mainly by range of proton reactivity. ASIC- as well as non-ASIC–expressing nociceptors have highly distinct cutaneous targets, and only one class was consistent with the existence of a generic C polymodal nociceptor (type 8a).