Sirko, P; (2015) ATP-activated currents and calcium signalling in adult murine supporting cells. Doctoral thesis , UCL (University College London).

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Abstract

Purinergic P2 receptors are present in the adult organ of Corti however their role and cellular effects of their stimulation are unclear. To date studies have been hampered by the relative difficulty of accessing intact adult organ of Corti tissue. In this study a novel dissection technique which enabled easy access to the apical turn of the adult mouse organ of Corti was used. The goal of the current study was to determine the distribution of P2 receptors and determine the effect of ATP on membrane conductances in inner sulcus (IS) supporting cells and supporting cells surrounding the IHC afferent synapse in the adult mouse organ of Corti. Purinergic P2 receptors were localized immunohistochemically and functionally to multiple supporting cells of the organ of Corti. In patch clamp electrophysiology recordings application of extracellular ATP to gap junction coupled IS cells, elicited inward currents and transiently increased resting membrane resistance while making the resting zero current potential more positive. Transmitted brightfield imaging showed that the electrophysiological changes were accompanied by a decrease in cytoplasmic Brownian movement. In IS cells decoupled with gap junction blockers stimulation with ATP elicited monophasically declining membrane currents. In contrast in decoupled border cells surrounding the IHC afferent synapse ATP activated biphasic currents. The initial component of the currents elicited in border cells desensitized during sustained stimulation and showed high inward rectification, characteristic of some P2X receptor subtypes. In contrast the delayed component was identified as a Ca2+-activated Cl- channel. It is argued that these conductances could help decrease the potassium concentration around the IHCs during periods of sustained IHC stimulation and thus affect the membrane potential of these sensory cells. In addition, actively propagating, gap junction-mediated, extracellular ATP-independent Ca2+ waves were observed to propagate along the organ of Corti in the IS and Deiters’ cell regions. These increases in intracellular Ca2+ levels were periodic. Currently the role of the waves is unclear, however increases in intracellular Ca2+ of a similar frequency and periodicity have been linked to the regulation of gene expression. Thus the observed waves could be a new mechanism regulating gene expression in the organ of Corti.

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