Probing the Composition of TMEM16A-Containing Signaling Complexes in Sensory Neurons

Abstract

TMEM16A (ANO1) is a Ca2+ activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons responding to painful (noxious) stimuli; these neurons also express the noxious heat sensor TRPV1. Our previous findings indicate that in these neurons, TMEM16A is specifically coupled to Ca2+ release from intracellular stores due to close association of TMEM16A channels, G protein coupled receptors, and IP3 receptors in multiprotein complexes assembled at plasma membrane-endoplasmic reticulum (PM-ER) junctions. Like TRPV1, TMEM16A is activated by noxious heat and functional coupling between the two channels has also been shown. We used live confocal imaging of Cl- channel activity, proximity ligation and super-resolution microscopy to investigate physical and functional relationships between TMEM16A and TRPV1 channels, and IP3 receptors in rat dorsal root ganglion (DRG) neurons. (i) Simultaneous monitoring of Cl- channel activity and Ca2+ dynamics in DRG neurons transfected with a halide-sensitive EYFP mutant (H148Q/I152L) and loaded with fura-2 revealed that the TRPV1 ligand, capsaicin, can robustly activate CaCC. However, ER store depletion significantly reduced (but not abolished) the capsaicin-induced Ca2+ rises and CaCC activation, suggesting that Ca2+ release from the ER contributes significantly to TRPV1-mediated CaCC activation. (ii) Proximity ligation assays established that in DRG neurons, TMEM16A and TRPV1 is often in close (less than 40 nm) proximity; similarly, close proximity between TMEM16A and IP3R1 was also observed. (iii) Stochastic optical reconstruction (STORM) microscopy confirmed close association of TMEM16A and TRPV1 channels in DRG neurons, representing 21% of the total population of labeled channels. We also observed close proximity of TMEM16A and IP3R1, and between TRPV1 and IP3R1, within 100 nm, although these molecules could be somewhat further apart, compared to membrane-localized TMEM16A/TRPV1 complexes, consistent with the ER membrane localization of IP3R1. Taken together, our data reveal the composition of TMEM16A-containing signaling complexes in DRG neurons and suggest that coupling between TRPV1 and TMEM16A requires ER Ca2+ release, which may be necessary to boost activation of TMEM16A, as this channel has a relatively low Ca2+ affinity.