FLUORESCENCE IMAGING OF NA-CELLS( INFLUX VIA P2X RECEPTORS IN COCHLEAR HAIR)

The adenosine 5'-triphosphate (ATP)-activated membrane conductance, me diated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elict ed a 30-ms latency inwardly rectifying nonselective cation conductance , blocked by the P2X receptor antagonist pyridoxalphosphate-6-azopheny l-2',4'-disulphonic acid (PPADS; 10-100 mu M), consistent with the dir ect activation of ATP-gated ion channels. A K-(Ca) conductance in the inner hair cells (IHC), activated by the entry of Ca2+ through the ATP -gated ion channels, was blocked by including 10 mM ,2-bis(2-aminophen oxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the internal solution . Real-time confocal slit-scanning fluorescence imaging of Na+ influx through the ATP-gated ion channels was performed using the dye Sodium Green(TM) with simultaneous whole-cell recording of membrane currents. The Na+ entry was localized to the endolymphatic surface, with the in crease in [Na+](i) detected within approximately 200 ms of the onset o f the inward current response. Within 600 ms Na+ had diffused througho ut the cell cytoplasm with the exception of the subnuclear region of t he outer hair cells. Correlation of voltage-clamp measurements of Naentry with regional increases in Naf-induced fluorescence demonstrated ATP-induced increases in intracellular Na+ in excess of 45 mM within 4 s. These data provide direct evidence for the Na+ permeability of th e ATP-gated ion channels as well as independent evidence for the local ization of P2X receptors at the endolymphatic surface of the sensory h air cells. The localization of the ATP-gated ion channels to the apica l surface of the hair cells supports an ATP-mediated modulation of 'si lent' K+ current across the cochlear partition which could regulate he aring sensitivity by controlling the transcellular driving force for b oth mechanoelectrical and electromechanical transduction in hair cells . (C) 1998 Elsevier Science B.V.