ABSTRACT
In the cochlea, Reissner's membrane separates the scala media endolymphatic compartment that sustains the positive endocochlear potential and ion composition necessary for sound transduction, from the scala vestibuli perilymphatic compartment. It is known that with sustained elevated sound levels, adenosine 5'-triphosphate (ATP) is released into the endolymph and ATP-gated ion channels on the epithelial cells lining the endolymphatic compartment shunt the electrochemical driving force, contributing to protective purinergic hearing adaptation. This study characterises the properties of epithelial cell P2X(2)-type ATP-activated membrane conductance in the mouse Reissner's membrane, which forms a substantial fraction of the scale media surface. The cells were found to express two isoforms (a and b) of the P2X(2) subunit arising from alternative splicing of the messenger RNA (mRNA) transcript that could contribute to the trimeric subunit assembly. The ATP-activated conductance demonstrated both immediate and delayed desensitisation consistent with incorporation of the combination of P2X(2) subunit isoforms. Activation by the ATP analogue 2meSATP had equipotency to ATP, whereas α,ß-meATP and adenosine 5'-diphosphate (ADP) were ineffective. Positive allosteric modulation of the P2X(2) channels by protons was profound. This native conductance was blocked by the P2X(2)-selective blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and the conductance was absent in these cells isolated from mice null for the P2rX2 gene encoding the P2X(2) receptor subunit. The activation and desensitisation properties of the Reissner's membrane epithelial cell ATP-gated P2X(2) channels likely contribute to the sensitivity and kinetics of purinergic control of the electrochemical driving force for sound transduction invoked by noise exposure.
Subject(s)
Adenosine Triphosphate/physiology , Cochlea/metabolism , Epithelial Cells/metabolism , Ion Channels/metabolism , Receptors, Purinergic P2X2/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Hearing , Ion Channels/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Purinergic P2X Receptor Antagonists/pharmacology , Pyridoxal Phosphate/analogs & derivatives , Pyridoxal Phosphate/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Purinergic P2X2/drug effects , Receptors, Purinergic P2X2/genetics , Thionucleotides/pharmacologyABSTRACT
Ryanodine receptor (RyR)-gated Ca2+ stores have recently been identified in cochlear spiral ganglion neurons (SGN) and likely contribute to Ca2+ signalling associated with auditory neurotransmission. Here, we identify an ionotropic glutamate receptor signal transduction pathway which invokes RyR-gated Ca2+ stores in SGN via Ca2+-induced Ca2+ release (CICR). Ca2+ levels were recorded in SGN in situ within rat cochlear slices (postnatal day 0-17) using the Ca2+ indicator fluo-4. RyR-gated Ca2+ stores were confirmed by caffeine-induced increases in intracellular Ca2+ which were blocked by ryanodine (100 microM) and were independent of external Ca2+. Glutamate evoked comparable increases in intracellular Ca2+, but required the presence of external Ca2+. Ca2+ influx via the glutamate receptor was found to elicit CICR via RyR-gated Ca2+ stores, as shown by the inhibition of the response by prior depletion of the Ca2+ stores with caffeine, the SERCA inhibitor thapsigargin, or ryanodine. The glutamate analogue AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) elicited Ca2+ responses that could be inhibited by caffeine. Glutamate- and AMPA-mediated Ca2+ responses were eliminated with the AMPA/Kainate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). These data demonstrate functional coupling between somatic AMPA-type glutamate receptors and intracellular Ca(2+) stores via RyR-dependent CICR in primary auditory neurons.