Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca²⁺ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea

Abstract

Using microfluidics, ATP biosensors, multiphoton microscopy and genetically targeted mice, we show that ATP release through connexin hemichannels, and not pannexin 1 channels, underlies spontaneous Ca2+ wave propagation in the greater epithelial ridge of the developing cochlea.

The Cl--channel TMEM16A controls the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks

“…Spontaneous Ca 2+ waves are elicited by ATP-induced ATP release from ISCs (Mazzarda, D’Elia et al 2020), a mechanism possibly involving P2Y1, P2Y2 and P2Y4 receptors (Piazza, Ciubotaru et al 2007, Babola, Li et al 2018), Cx26 and Cx30 heteromeric hemichannels (Anselmi, Hernandez et al 2008, Schutz, Scimemi et al 2010) and TMEM16A (Wang, Lin et al 2015). To assess what role TMEM16A might play in ATP-mediated Ca 2+ signals we applied the non-selective P2 receptor antagonist suramin (150 μM) (von Kugelgen and Wetter 2000, Burnstock 2014).…”

“…Since suramin blocked Ca 2+ waves in wildtype cochleae but had no effect on local Ca 2+ transients in cKO mice, the effect of TMEM16A on the propagation of Ca 2+ transients along Kölliker’s organ clearly involves the activation of purinergic receptors. ATP release by ISCs seems to occur through the opening of connexin hemichannels triggered by Cl - efflux-mediated osmotic cell shrinkage (Mazzarda, D’Elia et al 2020). Given the presence of mechanically induced ATP release and purinergic receptor activation in the generation of Ca 2+ waves in other tissues expressing TMEM16A such as the airway and submandibular gland epithelium (Sanderson, Charles et al 1990, Felix, Woodruff et al 1996, Grygorczyk and Hanrahan 1997, Watt, Lazarowski et al 1998, Homolya, Watt et al 1999, Homolya, Steinberg et al 2000, Romanenko, Catalan et al 2010, Ryu, Peixoto et al 2010), the olfactory epithelium (Hegg, Irwin et al 2009, Henriques, Agostinelli et al 2019), and the biliary epithelium (Dutta, Woo et al 2013), it will be interesting to investigate whether ATP/TMEM16A-mediated propagation of Ca 2+ waves is a wide-spread feature in many tissues.…”

“…In the developing inner ear, non-sensory inner supporting cells (ISCs) form a transient epithelial structure known as Kölliker’s organ (Hinojosa 1977, Hou, Chen et al 2019). ATP released from ISCs through connexin hemichannels (Mazzarda, D’Elia et al 2020) activates purinergic receptors in a paracellular manner leading to cell volume decrease of ISCs and cochlear Ca 2+ transients (Tritsch, Yi et al 2007, Tritsch and Bergles 2010, Babola, Li et al 2018). It was proposed that the Ca 2+ -activated Cl - -channel TMEM16A, which is expressed in ISCs, might be the pacemaker for spontaneous cochlear activity (Yi, Lee et al 2013).…”

Calcium signaling in the photodamaged skin

“…Expected band sizes were 450 bp for the CAG-GCamp6 and 297 bp for the wt allele and 390bp for the Cre transgene. To carry out this experimental work, we used a custom-made multiphoton system (Figure 1) based on a Bergamo II architecture (Thorlabs Imaging System, Sterling, VI, USA), as previously described [29]. The system was equipped with two scanning heads, one with resonant-galvo (RG) mirrors and the other with galvo-galvo (GG) mirrors, and was coupled to a mode-locked titanium-sapphire (Ti:Sa) fs pulsed laser (Chameleon Vision II Laser, Coherent, Inc., Santa Clara, CA, USA) (Figure 2).…”

“…In different epithelia, including epidermis, damage triggers perturbations of the [Ca 2+ ]c that spread from cell to cell (known as intercellular Ca 2+ waves) [25][26][27][28][29][30]. Ca 2+ waves are considered a fundamental mechanism for coordinating multicellular responses [31], however the mechanisms underlying Ca 2+ wave propagation and their significance in the damaged epidermis are incompletely understood.…”