Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells

Br J Pharmacol. 1997 Dec;122(7):1323-32. doi: 10.1038/sj.bjp.0701518.


1. The possible role of intracellular Ca2+ levels ([Ca2+]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 [Ca2+]i rises mainly due to influx through nicotinic AChRs. These responses were blocked by (+)-tubocurarine (10 microM) but were insensitive to alpha-bungarotoxin (1 microM) or Cd2+ (0.1 mM). 3. Pressure applications of 1 mM 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 [Ca2+]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 [Ca2+]i transient induced by the conditioning pulse. 4. Omission of intracellular Ca2+ chelators or use of high extracellular Ca2+ solution (10 mM) 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 Ca2+ buffers. None of these manipulations of external or internal Ca2+ had any effect on onset or steady state level of desensitization. 5. High spatial resolution imaging of [Ca2+]i in intact cells (in the presence of 0.1 mM Cd2+) showed that its level in the immediate submembrane area decayed at the same rate as in the rest of the cell, indicating that Ca2+ 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 [Ca2+]i and that this phenomenon retards their recovery to full activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Calcium / physiology
  • Chromaffin Cells / drug effects*
  • Chromaffin Cells / physiology
  • Female
  • Male
  • Membrane Potentials / drug effects
  • Microscopy, Confocal
  • Nicotine / pharmacology
  • Nicotinic Agonists / pharmacology*
  • Rats
  • Receptors, Nicotinic / drug effects*
  • Receptors, Nicotinic / metabolism
  • Tubocurarine / pharmacology


  • Nicotinic Agonists
  • Receptors, Nicotinic
  • Nicotine
  • Calcium
  • Tubocurarine