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Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells
Author(s) -
Khiroug L.,
Giniatullin R.,
Sokolova Elena,
Talantova Maria,
Nistri A.
Publication year - 1997
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0701518
Subject(s) - bapta , nicotinic agonist , biophysics , chemistry , nicotine , acetylcholine , intracellular , desensitization (medicine) , extracellular , egta , calcium in biology , endocrinology , receptor , medicine , calcium , biology , biochemistry , organic chemistry
1 The possible role of intracellular Ca 2+ levels ([Ca 2+ ] i ) in desensitization of nicotinic acetylcholine receptors (AChRs) was investigated in rat cultured chromaffin cells by use of combined whole‐cell patch clamping and confocal laser scanning microscopy with the fluorescent dye fluo‐3. 2 On cells held at −70 mV, pressure‐application of nicotine elicited inward currents with associated [Ca 2+ ] i rises mainly due to influx through nicotinic AChRs. These responses were blocked by (+)‐tubocurarine (10 μ M ) but were insensitive to α‐bungarotoxin (1 μ M ) or Cd 2+ (0.1 m M ). 3 Pressure applications of 1 m M nicotine for 2 s (conditioning pulse) evoked inward currents which faded biexponentially to a steady state level due to receptor desensitization and were accompanied by a sustained increase in [Ca 2+ ] i . Inward currents evoked by subsequent application of brief test pulses of nicotine were depressed but recovered with a time course reciprocal to the decay of the [Ca 2+ ] i transient induced by the conditioning pulse. 4 Omission of intracellular Ca 2+ chelators or use of high extracellular Ca 2+ solution (10 m M ) lengthened recovery of nicotinic AChRs from desensitization while adding BAPTA or EGTA intracellularly had the opposite effect. When the patch pipette contained fluo‐3 or no chelators, after establishing whole cell conditions the rate of recovery became progressively longer presumably due to dialysis of endogenous Ca 2+ buffers. None of these manipulations of external or internal Ca 2+ had any effect on onset or steady state level of desensitization. 5 High spatial resolution imaging of [Ca 2+ ] i in intact cells (in the presence of 0.1 m M Cd 2+ ) showed that its level in the immediate submembrane area decayed at the same rate as in the rest of the cell, indicating that Ca 2+ was in a strategic location to modulate (directly or indirectly) AChR desensitization. 6 The present data suggest that desensitized nicotinic AChRs are stabilized in their conformation by raised [Ca 2+ ] i and that this phenomenon retards their recovery to full activity.