We report that ANO1 (also known as TMEM16A) Ca 2+ -activated Cl − channels in small neurons from dorsal root ganglia are preferentially activated by particular pools of intracellular Ca 2+ . These ANO1 channels can be selectively activated by the G protein-coupled receptor (GPCR)-induced release of Ca 2+ from intracellular stores, but not by Ca 2+ influx through voltage-gated Ca 2+ channels. This ability to discriminate between Ca 2+ pools was achieved by the tethering of ANO1-containing plasma membrane domains, which also contained GPCRs such as bradykinin receptor-2 and protease-activated receptor-2, to juxtamembrane regions of the endoplasmic reticulum. Interaction of the C-terminus and the first intracellular loop of ANO1 with IP 3 R1 (inositol 1,4,5-trisphosphate receptor 1) contributed to the tethering. Disruption of membrane microdomains blocked the ANO1 and IP 3 R1 interaction and resulted in the loss of coupling between GPCR signaling and ANO1. The junctional signaling complex enabled ANO1-mediated excitation in response to specific Ca 2+ signals rather than to global changes in intracellular Ca 2+ .

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ANO1 (TMEM16A) is a Ca2+-activated Cl− channel (CaCC) expressed in peripheral somatosensory neurons that are activated by painful (noxious) stimuli. These neurons also express the Ca2+-permeable channel and noxious heat sensor TRPV1, which can activate ANO1. Here, we revealed an intricate mechanism of TRPV1-ANO1 channel coupling in rat dorsal root ganglion (DRG) neurons. Simultaneous optical monitoring of CaCC activity and Ca2+ dynamics revealed that the TRPV1 ligand capsaicin activated CaCCs. However, depletion of endoplasmic reticulum (ER) Ca2+ stores reduced capsaicin-induced Ca2+ increases and CaCC activation, suggesting that ER Ca2+ release contributed to TRPV1-induced CaCC activation. ER store depletion by plasma membrane–localized TRPV1 channels was demonstrated with an ER-localized Ca2+ sensor in neurons exposed to a cell-impermeable TRPV1 ligand. Proximity ligation assays established that ANO1, TRPV1, and the IP3 receptor IP3R1 were often found in close proximity to each other. Stochastic optical reconstruction microscopy (STORM) confirmed the close association between all three channels in DRG neurons. Together, our data reveal the existence of ANO1-containing multichannel nanodomains in DRG neurons and suggest that coupling between TRPV1 and ANO1 requires ER Ca2+ release, which may be necessary to enhance ANO1 activation.

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca 2+ ), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca 2+ channels which give rise to these Ca 2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca 2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca 2+ channels that are locally activated during the genesis of cytoplasmic Ca 2+ signals.

Rat somatosensory neurons express a junctional protein, junctophilin-4 (JPH4) r JPH4 is necessary for the formation of store operated Ca 2+ entry (SOCE) complex at the junctions between plasma membrane and endoplasmic reticulum in these neurons. r Knockdown of JPH4 impairs endoplasmic reticulum Ca 2+ store refill and junctional Ca 2+ signalling in sensory neurons. r In vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly attenuated experimentally induced inflammatory pain in rats. r Junctional nanodomain Ca 2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

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Junctions of endoplasmic reticulum and plasma membrane (ER-PM junctions) serve as signaling hubs in prokaryotic cells. ER-PM junctions are present in peripheral sensory neurons and are necessary for pro-inflammatory G protein coupled receptor signalling and for inflammatory pain generation. Yet, the principles of ER-PM junctions assembly and maintenance, as well as their role in inflammatory signaling in sensory neurons are only beginning to emerge. Here we discovered that a member of the junctophilin family of proteins, JPH4, is abundantly expressed in rat dorsal root ganglion (DRG) neurons and is necessary for the formation of store operated Ca 2+ entry (SOCE) complex at the ER-PM junctions in response to the G-protein induced ER Ca 2+ store depletion. Furthermore, we demonstrate a key role of the JPH4 and ER Ca 2+ stores in the maintenance of inflammatory pain. Indeed, knockdown of JPH4 expression in DRG in vivo significantly reduced the duration of pain produced by inflammatory mediator bradykinin. Since the ER supplies Ca 2+ for the excitatory action of multiple inflammatory mediators, we suggest that junctional Ca 2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

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