Intracellular calcium regulation in inner hair cells from neonatal mice.
The mechanisms that regulate the concentration of ionized intracellular calcium (Ca(2+)(i)) in the base of neonatal mouse inner hair cells, close to synaptic sites, were investigated using confocal microscopy combined with conventional patch-clamp electrophysiology. Cells were depolarized under whole-cell voltage clamp to load the cell with C a(2+) through voltage-activated Ca(2+) channels. Repeated depolarizations produced Ca(2+)(i) increases with similar amplitudes and time-courses of recovery. The rate of recovery from depolarization-induced Ca(2+)(i) loads was used to assess the mechanisms responsible for Ca(2+)(i) regulation. Removal of extracellular sodium had no effect on resting Ca(2+)(i) or the rate of recovery of Ca(2+)(i) suggesting no role for Na:Ca exchange in these cells. Inhibitors of intracellular store uptake such as thapsigargin, 2,5-di(tert-butyl)hydroquinone (BHQ) and cyclopiazonic acid (CPA) caused an increase in resting Ca(2+)(i) and slowed the rate of recovery, indicating that Ca(2+) can be taken up intracellularly. However, 5mM caffeine failed to cause a detectable release of Ca(2+) from intracellular stores. FCCP, a mitochondrial inhibitor, slowed the rate of recovery from Ca(2+)(i) loads, indicating a role for mitochondrial Ca(2+) uptake. The largest effects were seen with intracellular vanadate (1mM) which caused an irreversible rise in resting Ca(2+)(i) and depolarization-induced increases in Ca(2+)(i) failed to recover fully. Together, these data indicate that both thapsigargin-sensitive stores and mitochondria can take up Ca(2+)(i), but that Ca(2+) efflux from the cell occurs solely via a plasma membrane Ca(2+)-ATPase.
Kennedy
H J
HJ
Department of Physiology, School of Medical Sciences, University of Bristol, UK. Helen.Kennedy@bristol.ac.uk
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006199:Hair Cells, Auditory, Inner / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006706:Homeostasis; D006873:Hydroquinones / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D017136:Ion Transport; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006873:Hydroquinones; D007211:Indoles; D014966:Xanthenes; C059715:Fluo-3; D002110:Caffeine; D019284:Thapsigargin; D012964:Sodium; D002118:Calcium; C000543:cyclopiazonic acid