title: Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells.
abstract: 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.
authors.affiliation: Biophysics Sector and INFM Unit, International School of Advanced Studies (SISSA), Trieste, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Khiroug
authors.forename: R
authors.initials: R
authors.lastname: Giniatullin
authors.forename: E
authors.initials: E
authors.lastname: Sokolova
authors.forename: M
authors.initials: M
authors.lastname: Talantova
authors.forename: A
authors.initials: A
authors.lastname: Nistri
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D019439:Chromaffin Cells / Q000187:drug effects* / Q000502:physiology; D005260:Female; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology*; D051381:Rats; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000378:metabolism; D014403:Tubocurarine / Q000494:pharmacology
chemical_list: D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D009538:Nicotine; D002118:Calcium; D014403:Tubocurarine