Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog.
Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]i) were monitored optically in hair cells mechanically isolated from frog semicircular canals using the membrane-impermeant form of the Ca(2+)-selective dye Oregon Green 488 BAPTA-1 (OG, 100 microM). Cells stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca(2+) entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the cell body. [Ca(2+)]i at individual hotspots rose with a time constant tau1 approximately 70 ms and decayed with a bi-exponential time-course (tau2 approximately 160, tau3 approximately 2500 ms) following a 160 ms depolarization to -20 mV. With repeated stimulation [Ca(2+)]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca(2+) channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca(2+)channels are distributed at different densities in distinct hotspots. No diffusion barrier other than the nuclear region was found in the cytosol, so that, during a prolonged depolarization (lasting up to 1s), Ca(2+) was able to reach the cell apical ciliated pole. The effective Ca(2+) diffusion constant, measured from the progression of Ca(2+) wavefronts in the cytosol, was approximately 57 microm(2)/s. Our results indicate that in these hair cells, buffered diffusion of Ca(2+) proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers.
Rispoli
G
G
Dipartimento di Biologia e Istituto Nazionale di Fisica della Materia, Sezione di Fisiologia e Biofisica, Università di Ferrara, via Borsari, 46, Ferrara, 44100, Italy.
Martini
M
M
Rossi
M L
ML
Mammano
F
F
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006198:Hair Cells, Auditory / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D009930:Organic Chemicals; D018408:Patch-Clamp Techniques; D011893:Rana esculenta; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D005456:Fluorescent Dyes; C403124:Oregon Green BAPTA-dextran; D009930:Organic Chemicals; D009543:Nifedipine; D002118:Calcium