Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice

被引:167
作者
Woodruff, ML
Sampath, AP
Matthews, HR
Krasnoperova, NV
Lem, J
Fain, GL [1 ]
机构
[1] Univ Calif Los Angeles, Dept Physiol Sci, Los Angeles, CA 90095 USA
[2] Univ Calif Los Angeles, Dept Ophthalmol, Los Angeles, CA 90095 USA
[3] Univ Washington, Dept Physiol & Biophys, Seattle, WA 98195 USA
[4] Univ Cambridge, Physiol Lab, Cambridge CB2 3EG, England
[5] Tufts Univ New England Med Ctr, Genet Program, Dept Ophthalmol, Boston, MA 02111 USA
[6] Tufts Univ New England Med Ctr, Genet Program, Mol Cardiol Res Inst, Boston, MA 02111 USA
[7] Tufts Univ, Sch Med, Tufts Ctr Vis Res, Boston, MA 02111 USA
来源
JOURNAL OF PHYSIOLOGY-LONDON | 2002年 / 542卷 / 03期
关键词
D O I
10.1113/jphysiol.2001.013987
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
A 10 mum spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo-3, fluo-4 or fluo-5F, to estimate dark, resting free Ca2+ concentration ([Ca2+](i)) and changes in [Ca2+](i) upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild-type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin a-subunit knock-out (Tralpha-/-) animals showed no light-dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (tau similar to 1-4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre-exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca2+](i). A similar rapid increase in fluorescence was also seen in the rods of wild-type mice just preceding the fall in fluorescence produced by the light-dependent decrease in [Ca2+](i). Dissociation constants were measured in vitro for fluo-3, fluo-4 and fluo-5F with and without 1 mm Mg2+ from 20 to 37degreesC. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3-4 over this range. Values at 37degreesC were used to estimate absolute levels of rod [Ca2+](i). All three dyes gave similar values for [Ca2+](i) in wild-type rods of 250 +/- 20 nm in darkness and 23 +/- 2 nm after exposure to saturating light. There was no significant difference in dark [Ca2+](i) between wildtype and Tralpha-/- animals.
引用
收藏
页码:843 / 854
页数:12
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