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 μM) but were insensitive to α-bungarotoxin (1 μM) 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.

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