Regulation of the sensitivity of acetylcholine receptors to nicotine in rat habenula neurons
John H. Hicks, John A. Dani* and Robin A. J. Lester
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294 and *Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Time-dependent changes in nicotinic acetylcholine receptor (nAChR) function were studied in acutely isolated medial habenula neurons during whole-cell perfusion.
The peak amplitude of inward currents induced by 1 s pulses of nicotinic agonists, applied at 30 s intervals, gradually increased over the first several minutes of whole-cell recording. The ratio of response amplitudes at 1 and 15 min (t15/t1) was 1·9. Run-up of responses occurred independently of channel activation and was specific to nAChRs.
The channel blocker chlorisondamine (30 µM), co-applied with nicotine, was used to irreversibly block the majority (91 %) of the nAChRs that opened in the first 2 min of recording. Run-up in the remaining 9 % unblocked channels assessed at 15 min (t15/t2 = 3·4) was similar to that in control cells not exposed to nicotine and chlorisondamine simultaneously, implying that run-up is not due to the incorporation of new receptors.
A marked alteration in the sensitivity of nAChRs to extracellular Ca2+ was also observed during whole-cell perfusion. The ratio of current amplitudes obtained in 0·2 and 4·0 mM Ca2+ changed from 0·54 (t = 5 min) to 0·82 (t = 30 min).
Inward rectification of nicotine-induced responses was reduced during internal dialysis. Voltages for half-maximal conductance were -23·0 and -13·8 mV at 2 and 15 min, respectively. Inclusion of either free Mg2+ (~2 mM) or spermine (100 µM) in the internal solution counteracted the change in rectification, but did not prevent run-up.
The period of run-up was followed by a use-dependent run-down phase. Little run-down in peak current amplitude was induced provided that agonist was applied infrequently (5 min intervals), whereas applications at 30 s intervals produced a loss of channel function after ~15 min whole-cell perfusion. The time at which run-down began (~5-30 min) was correlated with the initial rate of nAChR desensitization (~200-4000 ms); slowly desensitizing nicotinic currents demonstrated delayed run-down.
We suggest that run-up of nAChR-mediated responses does not require receptor activation and may result from a change in channel open probability. We also hypothesize that channel run-down reflects accumulation of nAChRs in long-lived desensitized/inactivated states.
