Fluorescence imaging of Na+ influx via P2X receptors in cochlear hair cells.
The adenosine 5'-triphosphate (ATP)-activated membrane conductance, mediated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elicted a 30-ms latency inwardly rectifying non-selective cation conductance, blocked by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10-100 microM), consistent with the direct 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 1,2-his(2-aminophenoxy)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 with simultaneous whole-cell recording of membrane currents. The Na+ entry was localized to the endolymphatic surface, with the increase in [Na+]i detected within approximately 200 ms of the onset of the inward current response. Within 600 ms Na+ had diffused throughout the cell cytoplasm with the exception of the subnuclear region of the outer hair cells. Correlation of voltage-clamp measurements of Na+ entry with regional increases in Na+-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 the ATP-gated ion channels as well as independent evidence for the localization of P2X receptors at the endolymphatic surface of the sensory hair cells. The localization of the ATP-gated ion channels to the apical surface of the hair cells supports an ATP-mediated modulation of 'silent' K+ current across the cochlear partition which could regulate hearing sensitivity by controlling the transcellular driving force for both mechanoelectrical and electromechanical transduction in hair cells.
Housley
G D
GD
Department of Physiology, Faculty of Medicine and Health Science, University of Auckland, New Zealand. g.housley@auckland.ac.nz
Raybould
N P
NP
Thorne
P R
PR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D006199:Hair Cells, Auditory, Inner / Q000187:drug effects / Q000378:metabolism*; D018072:Hair Cells, Auditory, Outer / Q000187:drug effects / Q000378:metabolism*; D007473:Ion Channels / Q000187:drug effects* / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008027:Light; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D058848:Receptors, Purinergic P2X4; D012964:Sodium / Q000378:metabolism*
D005456:Fluorescent Dyes; D007473:Ion Channels; D009930:Organic Chemicals; D058919:Purinergic P2 Receptor Antagonists; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D058848:Receptors, Purinergic P2X4; C098118:sodium green; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D011188:Potassium; D002118:Calcium