Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution.
Signal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+)-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca(2+) signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction-diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (<1mu) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca(2+) buffers and Ca(2+)-ATPases. We finally show that experimental fluorescence data collected during Ca(2+) influx are not interpreted correctly if the [Ca(2+)](i) is estimated by assuming that Ca(2+) equilibrates instantly with its reactants. In our opinion, this approach is of potentially general interest as it can be easily adapted to the study of Ca(2+) dynamics in diverse biological systems.
Bortolozzi
Mario
M
Foundation for Advanced Biomedical Research, Venetian Institute of Molecular Medicine, via G. Orus 2, 35129 Padova, Italy.
Lelli
Andrea
A
Mammano
Fabio
F
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D020013:Calcium Signaling; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004058:Diffusion; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005453:Fluorescence; D006198:Hair Cells, Auditory / Q000502:physiology*; D018069:Hair Cells, Vestibular / Q000502:physiology*; D021962:Membrane Microdomains; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000502:physiology*; D011892:Rana catesbeiana; D009435:Synaptic Transmission
D015220:Calcium Channels; D002614:Chelating Agents; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium