School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UNITED KINGDOM.
ANO1 (TMEM16A) is a Ca2+ activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons responding to painful (noxious) stimuli. Previously, our lab has been able to demonstrate specific coupling of ANO1 to inositol 1,4,5- trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) via G-protein coupled receptor (GPCR) activation. This phenomenon underscores excitatory and noxious effects of some mediators of inflammatory pain, such as pro-algesic and vasoactive neuropeptide bradykinin.
To further investigate mechanisms of ANO1 activation in somatosensory neurons, I developed a dual imaging approach, which involved transfecting dorsal root ganglion (DRG) neurons with a halide sensitive EYFP mutant (H148Q/I152L) and simultaneous Ca2+ imaging to monitor CaCC activity. This methodology was successfully used to demonstrate robust coupling of CaCC activity to IP3R activation produced by bradykinin. Blockade of ANO1 using a selective inhibitor (T16A-inhA01) abolished CaCC activity induced by bradykinin application. In contrast to the ER- induced Ca2+ release, Ca2+ influx produced by depolarisation-induced activation of voltage gated Ca2+ channels (VGCCs) was relatively ineffective in activating ANO1, which is in good agreement with previous studies.
TRPV1 activation by capsaicin was able to induce robust CaCC activity. Given the ability of TRPV1 to activate PLC isoforms and produce IP3, I further tested the mechanism by which ANO1 is activated by TRPV1. Depletion of the ER Ca2+ stores severely reduced both, the capsaicin-induced Ca2+ signals and the concurrent CaCC activation. Intriguingly, under extracellular Ca2+ free conditions capsaicin was still able to induce [Ca2+]i elevation, further illustrating the ability of TRPV1 to induce intracellular Ca2+ release. Finally, monitoring of ER specific-Ca2+ dynamics concurrently with CaCC activity unambiguously confirmed the ability of TRPV1 to produce ER-Ca2+ mobilisation. Importantly, IP3R blockade with xestospongin C reduced CaCC activity after TRPV1 activation. Collectively, these experiments suggest that a significant fraction of Ca2+ required for activation of ANO1 downstream of TRPV1 is indeed delivered through IP3R activation.
Using ‘in-situ proteomics’ and super-resolution microscopy I investigated multi- protein complexes in ER-plasma membrane (ER-PM) junctions of DRG neurons involving ANO1, TRPV1 and IP3R1. I found using proximity ligation assay that all 3 proteins were within 40nm of each other; however there was a greater number of ANO1 and TRPV1 complexes compared to TRPV1/ANO1 and TRPV1/IP3R1 complexes. Two-colour stochastic optical reconstruction microscopy (STORM) was able to confirm these findings and demonstrate that there is indeed a greater percentage of complexes involving ANO1 and TRPV1. Preliminary triple-colour STORM suggested the presence of ANO1, TRPV1 and IP3R1-containing protein complexes.
Finally, I used total internal reflection microscopy (TIRF) to monitor the dynamics of the ER-PM junctions following the activation of bradykinin receptors or TRPV1. Application of bradykinin and capsaicin elicited increased intensity proximity of the ER to PM (as evaluated by the TIRF signal of fluorescently-labelled ER), which is suggestive of the ER moving to the PM by internal store mobilisation and highlighting the importance of ER-PM junctions.
In sum, the experiments described in this thesis have discovered and characterised a novel mode of ANO1 activation in pain-sensing neurons: TRPV1-mediated ER Ca2+ release in ER-PM junctional signalling complex. These findings describe a hitherto unknown signalling mechanism potentially contributing to inflammatory pain.